Amide based insulin prodrugs

ABSTRACT

Prodrug formulations of insulin and insulin analogs are provided wherein the insulin peptide has been modified by an amide bond linkage of a dipeptide prodrug element. The prodrugs disclosed herein have extended half lives of at least 10 hours, and more typically greater than 2 hours, 20 hours and less than 70 hours, and are converted to the active form at physiological conditions through a non-enzymatic reaction driven by chemical instability.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a U.S. national counterpart application ofinternational application serial No. PCT/US2009/068716 filed Dec. 18,2009, which claims priority to U.S. Provisional Patent Application No.61/139,218 filed Dec. 19, 2008. The entire disclosures ofPCT/US2009/068716 and U.S. Ser. No. 61/139,218 are hereby incorporatedby reference.

INCORPORATION BY REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY

Incorporated by reference in its entirety is a computer-readablenucleotide/amino acid sequence listing submitted concurrently herewithand identified as follows: One 34 KB ACII (Text) file named seqlistinsulin prodrug revised 1 22 10 ST25.txt created on May 2, 2011.

BACKGROUND

Insulin is a peptide hormone comprised of a two chain heterodimer thatis biosynthetically derived from a low potency single chain proinsulinprecursor through enzymatic processing. Human insulin is comprised oftwo peptide chains (an “A chain” (SEQ ID NO: 1) and “B chain” (SEQ IDNO: 2)) bound together by disulfide bonds and having a total of 51 aminoacids. The C-terminal region of the B-chain and the two terminal regionsof the A-chain associate in a three-dimensional structure to assemble asite for high affinity binding to the insulin receptor.

Insulin demonstrates unparalleled ability to lower glucose in virtuallyall forms of diabetes. Unfortunately, its pharmacology is not glucosesensitive and as such it is capable of excessive action that can lead tolife-threatening hypoglycemia. Inconsistent pharmacology is a hallmarkof insulin therapy such that it is extremely difficult to normalizeblood glucose without occurrence of hypoglycemia. Furthermore, nativeinsulin is of short duration of action and requires modification torender it suitable for use in control of basal glucose. Establishedapproaches to delay the onset of insulin action include reduction insolubility, and albumin binding.

For example, two commercially available insulin derivatives have beenprepared to provide a longer action profile. More particularly, theinsulin derivative [GlyA21, ArgB31, ArgB32]insulin was prepared to shiftinsulin's pI from 5.4 to 6.7 resulting in the peptide being precipitatedat physiological pH and thus delaying adsorption and time of action (seeBolli et al., Diabetologia 1999, 42, 1151-1167). However, this insulinderivative has enhanced IGF-1 affinity, leading to increasedproliferative actions and the possibility of tumorigenesis. Anothercommercially available insulin derivative is [LysB29-tetradecanoyl,des(B30)]insulin, wherein LysB29 has been acylated with a C₁₄ fatty acid(Mayer et al., Peptide Science, 88, 5, 687-713). The presence of thefatty acid chain enhances binding of the peptide to serum albumin,resulting in increased plasma half life. However, this derivativesuffers the disadvantage of having reduced potency in vivo. In addition,both insulin derivatives exhibit variability in biological action fromone patient to the next.

Prodrug chemistry offers the opportunity to precisely control the onsetand duration of insulin action after clearance from the site ofadministration and equilibration in the plasma at a highly definedconcentration. The central virtue of such an approach, relative tocurrent long-acting insulin analogs and formulations, is that theinsulin reservoir is not the subcutaneous fatty tissue where injectionoccurs, but rather the blood compartment. This removes the variabilityin absorption encountered with prior art delayed onset insulinderivatives. It also enables administration of the peptide hormone byroutes other than a subcutaneous injection.

Binding of insulin to its receptor will result in biologicalstimulation, but will also initiate the subsequent deactivation ofinsulin induced pharmacology through the enzymatic degradation of theinsulin peptide. An added advantage of using a prodrug derivative ofinsulin is that such an approach also extends insulin's biological halflife based on a strategy of inhibiting recognition of the prodrug by thecorresponding receptor. In spite of these advantages associated withprodrug derivatives, the complex nature of preparing such prodrugs has,until now, prevented the preparation of an efficacious prodrugderivative of insulin. To build a successful prodrug-hormone, an activesite structural address is needed that can form the basis for thereversible attachment of a prodrug structural element. The structuraladdress needs to offer two key features; (1) the potential for selectivechemical modification and (2) the ability to provide a high degree ofactivity in the native form upon removal of the prodrug structuralelement. The insulin prodrugs disclosed herein are chemically convertedto structures that can be recognized by the receptor, wherein the speedof this chemical conversion will determine the time of onset andduration of in vivo biological action. The prodrug chemistry disclosedin this application relies upon an intramolecular chemical reaction thatis not dependent upon additional chemical additives, or enzymes orenzyme inhibitors.

The ideal prodrug should be soluble in water at physiological conditions(for example, a pH of 7.2 and 37° C.), and it should be stable in thepowder form for long term storage. It should also be immunologicallysilent and exhibit a low activity relative to the parent drug. Typicallythe prodrug will exhibit no more than 10% of the activity of the parentdrug, in one embodiment the prodrug exhibits less than 10%, less than5%, about 1%, or less than 1% activity relative to the parent drug.Furthermore, the prodrug, when injected in the body, should bequantitatively converted to the active drug within a defined period oftime. Applicants are the first to disclose insulin prodrug analogs thatmeet each of these objectives.

SUMMARY

Peptide-based drugs are highly effective medicines with relatively shortduration of action and variable therapeutic index. The presentdisclosure is directed to insulin prodrugs wherein the prodrugderivative is designed to delay onset of action and extend the half lifeof the drug. The delayed onset of action is advantageous in that itallows systemic distribution of the prodrug prior to its activation.Accordingly, the administration of prodrugs eliminates complicationscaused by peak activities upon administration and increases thetherapeutic index of the parent drug.

In accordance with one embodiment, a prodrug derivative of insulin isprepared by covalently linking a dipeptide to an active site of theinsulin peptide via an amide linkage. In one embodiment the dipeptide iscovalently bound to the insulin peptide at a position that interfereswith insulin's ability to interact with the insulin and IGF-1 receptors.Subsequent removal of the dipeptide via an intramolecular reaction,resulting in diketopiperazine or diketomorpholine formation, underphysiological conditions and in the absence of enzymatic activity,restores full activity to the polypeptide.

In one embodiment an insulin prodrug is provided having the generalstructure of U-O-insulin, wherein U is an amino acid or a hydroxyl acidand O is an N-alkylated amino acid linked to the insulin peptide throughformation of an amide bond between U-O and an amine of an insulinpeptide. In one embodiment the U-O dipeptide is bound at the N-terminus,or at the side chain of an amino acid corresponding to positions A19,B16 or B25, of the respective A chain or B chain via an amide bond. Thestructure of U-O is selected, in one embodiment wherein chemicalcleavage of U-O from the insulin peptide is at least about 90% completewithin about 1 to about 720 hours in PBS under physiological conditions.In one embodiment the chemical cleavage half-life (t_(1/2)) of U-O fromthe insulin peptide is at least about 1 hour to about 1 week in PBSunder physiological conditions.

In one embodiment U and O are selected to inhibit enzymatic cleavage ofthe U-O dipeptide from an insulin peptide by enzymes found in mammalianserum. In one embodiment U and/or O are selected such that the cleavagehalf-life of U-O from the insulin peptide, in PBS under physiologicalconditions, is not more than two fold the cleavage half-life of U-O fromthe insulin peptide in a solution comprising a DPP-IV protease (i.e.,cleavage of U-O from the insulin prodrug does not occur at a rate morethan 2× faster in the presence of DPP-IV protease and physiologicalconditions relative to identical conditions in the absence of theenzyme). In one embodiment U, O, or the amino acid of the insulinpeptide to which U-O is linked is a non-coded amino acid. In oneembodiment U and/or O is an amino acid in the D stereoisomerconfiguration. In some exemplary embodiments, U is an amino acid in theD stereoisomer configuration and O is an amino acid in the Lstereoisomer configuration. In some exemplary embodiments, U is an aminoacid in the L stereoisomer configuration and O is an amino acid in the Dstereoisomer configuration. In some exemplary embodiments, U is an aminoacid in the D stereoisomer configuration and O is an amino acid in the Dstereoisomer configuration. In one embodiment O is an N-alkylated aminoacid but is not proline.

In one embodiment the dipeptide prodrug element comprises a compoundhaving the general structure of Formula I:

wherein

R₁, R₂, R₄ and R₈ are independently selected from the group consistingof H, C₁-C₁₈ alkyl, C₂-C₁₈ alkenyl, (C₁-C₁₈ alkyl)OH, (C₁-C₁₈ alkyl)SH,(C₂-C₃ alkyl)SCH₃, (C₁-C₄ alkyl)CONH₂, (C₁-C₄ alkyl)COOH, (C₁-C₄alkyl)NH₂, (C₁-C₄ alkyl)NHC(NH₂ ⁺)NH₂, (C₀-C₄ alkyl)(C₃-C₆ cycloalkyl),(C₀-C₄ alkyl)(C₂-C₅ heterocyclic), (C₀-C₄ alkyl)(C₆-C₁₀ aryl)R₇, (C₁-C₄alkyl)(C₃-C₉ heteroaryl), and C₁-C₁₂ alkyl(W)C₁-C₁₂ alkyl, wherein W isa heteroatom selected from the group consisting of N, S and O, or R₁ andR₂ together with the atoms to which they are attached form a C₃-C₁₂cycloalkyl or aryl; or R₄ and R₈ together with the atoms to which theyare attached form a C₃-C₆ cycloalkyl;

R₃ is selected from the group consisting of C₁-C₁₈ alkyl, (C₁-C₁₈alkyl)OH, (C₁-C₁₈ alkyl)NH₂, (C₁-C₁₈ alkyl)SH, (C₀-C₄alkyl)(C₃-C₆)cycloalkyl, (C₀-C₄ alkyl)(C₂-C₅ heterocyclic), (C₀-C₄alkyl)(C₆-C₁₀ aryl)R₇, and (C₁-C₄ alkyl)(C₃-C₉ heteroaryl) or R₄ and R₃together with the atoms to which they are attached form a 4, 5 or 6member heterocyclic ring;

R₅ is NHR₆ or OH;

R₆ is H, C₁-C₈ alkyl or R₆ and R₂ together with the atoms to which theyare attached form a 4, 5 or 6 member heterocyclic ring; and

R₇ is selected from the group consisting of H and OH.

In one embodiment the dipeptide prodrug element comprises a compoundhaving the general structure of Formula I:

wherein

R₁, R₂, R₄ and R₈ are independently selected from the group consistingof H, C₁-C₁₈ alkyl, C₂-C₁₈ alkenyl, (C₁-C₁₈ alkyl)OH, (C₁-C₁₈ alkyl)SH,(C₂-C₃ alkyl)SCH₃, (C₁-C₄ alkyl)CONH₂, (C₁-C₄ alkyl)COOH, (C₁-C₄alkyl)NH₂, (C₁-C₄ alkyl)NHC(NH₂ ⁺)NH₂, (C₀-C₄ alkyl)(C₃-C₆ cycloalkyl),(C₀-C₄ alkyl)(C₂-C₅ heterocyclic), (C₀-C₄ alkyl)(C₆-C₁₀ aryl)R₇, (C₁-C₄alkyl)(C₃-C₉ heteroaryl), and C₁-C₁₂ alkyl(W₁)C₁-C₁₂ alkyl, wherein W₁is a heteroatom selected from the group consisting of N, S and O, or R₁and R₂ together with the atoms to which they are attached form a C₃-C₁₂cycloalkyl; or R₄ and R₈ together with the atoms to which they areattached form a C₃-C₆ cycloalkyl;

R₃ is selected from the group consisting of C₁-C₁₈ alkyl, (C₁-C₁₈alkyl)OH, (C₁-C₁₈ alkyl)NH₂, (C₁-C₁₈ alkyl)SH, (C₀-C₄alkyl)(C₃-C₆)cycloalkyl, (C₀-C₄ alkyl)(C₂-C₅ heterocyclic), (C₀-C₄alkyl)(C₆-C₁₀ aryl)R₇, and (C₁-C₄ alkyl)(C₃-C₉ heteroaryl) or R₄ and R₃together with the atoms to which they are attached form a 4, 5 or 6member heterocyclic ring;

R₅ is NHR₆ or OH;

R₆ is H, C₁-C₈ alkyl or R₆ and R₁ together with the atoms to which theyare attached form a 4, 5 or 6 member heterocyclic ring; and

R₇ is selected from the group consisting of hydrogen, C₁-C₁₈ alkyl,C₂-C₁₈ alkenyl, (C₀-C₄ alkyl)CONH₂, (C₀-C₄ alkyl)COOH, (C₀-C₄ alkyl)NH₂,(C₀-C₄ alkyl)OH, and halo.

In one embodiment the dipeptide extension comprises a compound of thegeneral structure:

wherein R₁ is selected from the group consisting of H and C₁-C₈ alkyl;and

-   -   R₂ and R₄ are independently selected from the group consisting        of H, C₁-C₈ alkyl, C₂-C₈ alkenyl, (C₁-C₄ alkyl)OH, (C₁-C₄        alkyl)SH, (C₂-C₃ alkyl)SCH₃, (C₁-C₄ alkyl)CONH₂, (C₁-C₄        alkyl)COOH, (C₁-C₄ alkyl)NH₂, (C₁-C₄ alkyl)NHC(NH₂ ⁺) NH₂,        (C₀-C₄ alkyl)(C₃-C₆ cycloalkyl), (C₀-C₄ alkyl)(C₆-C₁₀ aryl)R₇,        and CH₂(C₅-C₉ heteroaryl), or R₁ and R₂ together with the atoms        to which they are attached form a C₃-C₈ cycloalkyl ring;    -   R₃ is selected from the group consisting of C₁-C₈ alkyl, (C₁-C₄        alkyl)OH, (C₁-C₄ alkyl)SH, (C₁-C₄ alkyl)NH₂, (C₃-C₆)cycloalkyl        or R₄ and R₃ together with the atoms to which they are attached        form a 5 or 6 member heterocyclic ring;    -   R₅ is NHR₆ or OH;    -   R₆ is H, or R₆ and R₂ together with the atoms to which they are        attached form a 5 or 6 member heterocyclic ring; and    -   R₇ is selected from the group consisting of H and OH. In one        embodiment R₃ is C₁-C₈ alkyl and R₄ is selected from the group        consisting of H, C₁-C₈ alkyl, (C₁-C₄ alkyl)OH, (C₁-C₄ alkyl)SH,        (C₂-C₃ alkyl)SCH₃, (C₁-C₄ alkyl)CONH₂, (C₁-C₄ alkyl)COOH, (C₁-C₄        alkyl)NH₂, (C₁-C₄ alkyl)NHC(NH₂ ⁺) NH₂, (C₀-C₄ alkyl)(C₃-C₆        cycloalkyl), (C₀-C₄ alkyl)(C₆-C₁₀ aryl)R₇, and CH₂(C₅-C₉        heteroaryl) or R₄ and R₃ together with the atoms to which they        are attached form a 5 or 6 member heterocyclic ring.

In accordance with one embodiment an insulin prodrug analog is providedcomprising an A chain and a B chain, wherein the A chain comprises thesequence Z-GIVEQCCX₁SICSLYQLENX₂CX₃ (SEQ ID NO: 3) and the B chaincomprises the sequence of J-X₁₄-X₄LCGX₅X₆LVEALX₇LVCG ERGFX₈ (SEQ ID NO:14). The Z and J designations of the A and B chain formulas areindependently H (forming an N-terminal amine) or a dipeptide comprisingthe general structure:

wherein

X₁₄ is either a bond joining the “J” element to the N-terminus of theX₄LCGX₅X₆LVEALX₇LVCG ERGFX₈ (SEQ ID NO: 14) sequence or X₁₄ represents a1 to 4 amino acid sequence selected from the group consisting of a FVNQ(SEQ ID NO: 11), VNQ, NQ and Q that joins the “J” element to theN-terminus of the X₄LCGX₅X₆LVEALX₇LVCG ERGFX₈ (SEQ ID NO: 14) sequence.

X₁ is selected from the group consisting of threonine and histidine;

X₂ is an amino acid of the general structure

-   -   wherein X is selected from the group consisting of OH, NH₂,        NHR₁₀ and OCH₃, wherein R₁₀ is a dipeptide comprising the        general structure:

X₃ is selected from the group consisting of asparagine, ornathine,glycine, alanine, threonine, and serine;

X₄ is selected from the group consisting of histidine and threonine;

X₅ is selected from the group consisting of alanine, glycine and serine;

X₆ is selected from the group consisting of histidine, aspartic acid,glutamic acid, homocysteic acid and cysteic acid;

X₇ is an amino acid of the general structure

-   -   wherein X₁₂ is selected from the group consisting of OH, NH₂,        NHR₁₁ and OCH₃, wherein R₁₁ is a dipeptide comprising the        general structure:

X₈ is an amino acid of the general structure

-   -   wherein X₁₃ is selected from the group consisting of H, OH, NH₂,        NHR₁₂ and OCH₃, wherein R₁₂ is a dipeptide comprising the        general structure:

-   -   wherein R₁ is selected from the group consisting of H and C₁-C₈        alkyl;    -   R₂ and R₄ are independently selected from the group consisting        of H, C₁-C₈ alkyl, C₂-C₈ alkenyl, (C₁-C₄ alkyl)OH, (C₁-C₄        alkyl)SH, (C₂-C₃ alkyl)SCH₃, (C₁-C₄ alkyl)CONH₂, (C₁-C₄        alkyl)COOH, (C₁-C₄ alkyl)NH₂, (C₁-C₄ alkyl)NHC(NH₂ ⁺) NH₂,        (C₀-C₄ alkyl)(C₃-C₆ cycloalkyl), (C₀-C₄ alkyl)(C₆-C₁₀ aryl)R₇,        and CH₂(C₅-C₉ heteroaryl);    -   R₃ is selected from the group consisting of C₁-C₈ alkyl, (C₁-C₄        alkyl)OH, (C₁-C₄ alkyl)SH, (C₃-C₆)cycloalkyl or R₄ and R₃        together with the atoms to which they are attached form a 5 or 6        member heterocyclic ring;    -   R₅ is NHR₆ or OH;    -   R₆ is H, or R₆ and R₂ together with the atoms to which they are        attached form a 5 or 6 member heterocyclic ring; and    -   R₇ is selected from the group consisting of H and OH, with the        proviso that one and only one of X, X₁₂, X₁₃, J and Z comprises        a dipeptide of the general structure:

In one embodiment when J or Z comprise the dipeptide of Formula I, andR₄ and R₃ together with the atoms to which they are attached form a 4, 5or 6 member heterocyclic ring, then both R₁ and R₂ are not hydrogen

In accordance with one embodiment the dipeptide present at Z, J, R₁₀,R₁₁ or R₁₂ comprises a compound having the general structure of FormulaI:

wherein

R₁, R₂, R₄ and R₈ are independently selected from the group consistingof H, C₁-C₁₈ alkyl, C₂-C₁₈ alkenyl, (C₁-C₁₈ alkyl)OH, (C₁-C₁₈ alkyl)SH,(C₂-C₃ alkyl)SCH₃, (C₁-C₄ alkyl)CONH₂, (C₁-C₄ alkyl)COOH, (C₁-C₄alkyl)NH₂, (C₁-C₄ alkyl)NHC(NH₂ ⁺)NH₂, (C₀-C₄ alkyl)(C₃-C₆ cycloalkyl),(C₀-C₄ alkyl)(C₂-C₅ heterocyclic), (C₀-C₄ alkyl)(C₆-C₁₀ aryl)R₇, (C₁-C₄alkyl)(C₃-C₉ heteroaryl), and C₁-C₁₂ alkyl(W₁)C₁-C₁₂ alkyl, wherein W₁is a heteroatom selected from the group consisting of N, S and O, or R₁and R₂ together with the atoms to which they are attached form a C₃-C₁₂cycloalkyl; or R₄ and R₈ together with the atoms to which they areattached form a C₃-C₆ cycloalkyl;

R₃ is selected from the group consisting of C₁-C₁₈ alkyl, (C₁-C₁₈alkyl)OH, (C₁-C₁₈ alkyl)NH₂, (C₁-C₁₈ alkyl)SH, (C₀-C₄alkyl)(C₃-C₆)cycloalkyl, (C₀-C₄ alkyl)(C₂-C₅ heterocyclic), (C₀-C₄alkyl)(C₆-C₁₀ aryl)R₇, and (C₁-C₄ alkyl)(C₃-C₉ heteroaryl) or R₄ and R₃together with the atoms to which they are attached form a 4, 5 or 6member heterocyclic ring;

R₅ is NHR₆ or OH;

R₆ is H, C₁-C₈ alkyl or R₆ and R₁ together with the atoms to which theyare attached form a 4, 5 or 6 member heterocyclic ring; and

R₇ is selected from the group consisting of hydrogen, C₁-C₁₈ alkyl,C₂-C₁₈ alkenyl, (C₀-C₄ alkyl)CONH₂, (C₀-C₄ alkyl)COOH, (C₀-C₄ alkyl)NH₂,(C₀-C₄ alkyl)OH, and halo.

In accordance with one embodiment the dipeptide present at Z, J, R₁₀,R₁₁ or R₁₂ comprises a compound having the general structure of FormulaI:

wherein

R₁ and R₈ are independently H or C₁-C₈ alkyl;

R₂ and R₄ are independently selected from the group consisting of H,C₁-C₈ alkyl, C₂-C₈ alkenyl, (C₁-C₄ alkyl)OH, (C₁-C₄ alkyl)SH, (C₂-C₃alkyl)SCH₃, (C₁-C₄ alkyl)CONH₂, (C₁-C₄ alkyl)COOH, (C₁-C₄ alkyl)NH₂,(C₁-C₄ alkyl)NHC(NH₂+) NH₂, (C₀-C₄ alkyl)(C₃-C₆ cycloalkyl), (C₀-C₄alkyl)(C₂-C₅ heterocyclic), (C₀-C₄ alkyl)(C₆-C₁₀ aryl)R₇, and CH₂(C₃-C₉heteroaryl), or R₁ and R₂ together with the atoms to which they areattached form a C₃-C₁₂ cycloalkyl;

R₃ is C₁-C₁₈ alkyl;

R₅ is NHR₆;

R₆ is H or C₁-C₈ alkyl; and

R₇ is selected from the group consisting of hydrogen, C₁-C₁₈ alkyl,C₂-C₁₈ alkenyl, (C₀-C₄ alkyl)CONH₂, (C₀-C₄ alkyl)COOH, (C₀-C₄ alkyl)NH₂,(C₀-C₄ alkyl)OH, and halo.

In accordance with one embodiment an insulin analog is provided whereinthe A chain of the insulin peptide comprises the sequenceZ-GIVEQCCTSICSLYQLENX₂CN (SEQ ID NO: 6) and the B chain comprising asequence selected from the group consisting of HLCGSHLVEALYLVCGERGFF(SEQ ID NO: 7), FVNQHLCGSHLVEALYLVCGERGFFYTPKT (SEQ ID NO: 8) andFVNQHLCGSHLVEALYLVCGERGFFYTKPT (SEQ ID NO: 9) wherein Z is H or adipeptide comprising the general structure:

X₂ is an amino acid of the general structure

wherein R₁₀ is H or a dipeptide comprising the general structure:

with the proviso that Z and R₁₀ are not both H and are not both adipeptide comprising the general structure:

In accordance with one embodiment single-chain insulin prodrug analogsare provided. In this embodiment the carboxy terminus of the humaninsulin B chain, or a functional analog thereof, is covalently linked tothe N-terminus of the human insulin A chain, or functional analogthereof, wherein a dipeptide prodrug moiety having the generalstructure:

is covalently bound at the N-terminus of the peptide or at the sidechain of an amino acid corresponding to positions A19, B16 or B25 of therespective A chain or B chain via an amide bond. In one embodiment the Bchain is linked to the A chain via peptide linker of 4-12 or 4-8 aminoacids.

In another embodiment the solubility of the insulin prodrug analogs isenhanced by the covalent linkage of a hydrophilic moiety to the peptide.In one embodiment the hydrophilic moiety is linked to either theN-terminal amino acid of the B chain or to the amino acid at position 28of SEQ ID NO: 9 or the amino acid at position 29 of SEQ ID NO: 8. In oneembodiment the hydrophilic moiety is a polyethylene glycol (PEG) chain,having a molecular weight selected from the range of about 500 to about40,000 Daltons. In one embodiment the polyethylene glycol chain has amolecular weight selected from the range of about 500 to about 5,000Daltons. In another embodiment the polyethylene glycol chain has amolecular weight of about 10,000 to about 20,000 Daltons.

Acylation or alkylation can increase the half-life of the insulinpeptides in circulation. Acylation or alkylation can advantageouslydelay the onset of action and/or extend the duration of action at theinsulin receptors upon activation of the prodrug. The insulin analogsmay be acylated or alkylated at the same amino acid position where ahydrophilic moiety is linked, or at a different amino acid position.

In accordance with one embodiment a pharmaceutical composition isprovided comprising any of the novel insulin prodrug analogs disclosedherein, preferably at a purity level of at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98% or 99%, and a pharmaceutically acceptablediluent, carrier or excipient. Such compositions may contain an A19insulin analog as disclosed herein at a concentration of at least 0.5mg/ml, 1 mg/ml, 2 mg/ml, 3 mg/ml, 4 mg/ml, 5 mg/ml, 6 mg/ml, 7 mg/ml, 8mg/ml, 9 mg/ml, 10 mg/ml, 11 mg/ml, 12 mg/ml, 13 mg/ml, 14 mg/ml, 15mg/ml, 16 mg/ml, 17 mg/ml, 18 mg/ml, 19 mg/ml, 20 mg/ml, 21 mg/ml, 22mg/ml, 23 mg/ml, 24 mg/ml, 25 mg/ml or higher. In one embodiment thepharmaceutical compositions comprise aqueous solutions that aresterilized and optionally stored contained within various packagecontainers. In other embodiments the pharmaceutical compositionscomprise a lyophilized powder. The pharmaceutical compositions can befurther packaged as part of a kit that includes a disposable device foradministering the composition to a patient. The containers or kits maybe labeled for storage at ambient room temperature or at refrigeratedtemperature.

In accordance with one embodiment an improved method of regulating bloodglucose levels in insulin dependent patients is provided. The methodcomprises the steps of administering an insulin prodrug analog of thepresent disclosure in an amount therapeutically effective for thecontrol of diabetes. In one embodiment the insulin prodrug analog ispegylated with a PEG chain having a molecular weight selected from therange of about 5,000 to about 40,000 Daltons

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. is a schematic overview of the two step synthetic strategy forpreparing human insulin. Details of the procedure are provided inExample 1.

FIG. 2 is a graph comparing insulin receptor specific binding ofsynthetic human insulin relative to purified native insulin. Asindicated by the data presented in the graph, the two molecules havesimilar binding activities.

FIG. 3 is a graph comparing relative insulin receptor binding of nativeinsulin and the A19 insulin analog (Insulin(p-NH₂—F)¹⁹). As indicated bythe data presented in the graph, the two molecules have similar bindingactivities.

FIG. 4 is a graph comparing relative insulin receptor binding of nativeinsulin and the IGF1(YL)^(B16B17) analog. As indicated by the datapresented in the graph, the two molecules have similar bindingactivities.

FIG. 5 is a schematic drawing of the synthetic scheme used to preparethe IGF1(Y^(B16)L¹⁷)(p-NH₂—F)^(A19) analog (will not be activated).

FIG. 6 is a graph comparing relative insulin receptor binding ofIGF1(Y^(B16)L^(B17))(p-NH₂—F)^(A19) and the dipeptide extended form ofIGF1(Y^(B16)L^(B17))(p-NH₂—F)^(A19), wherein the dipeptide AiBAla isbound at position A19 (i.e. IGF1(Y^(B16)L¹⁷)^(A19)-AiBAla).

FIG. 7A-7C provides the activity of a dimer prepared in accordance withthe present disclosure. FIG. 7A shows the structure of an IGF-1 singlechain dimer that comprises two single chain IGF^(B16B17) derivativepeptides (IGF-1B chain[C⁰H⁵Y¹⁶L¹⁷O²²]-A chain[O^(9,14,15)N^(18,21)]; SEQID NO: 68) linked together by a disulfide bond between the side chainsof the amino terminus of the B chains. FIG. 7B is a graph demonstratingthe relative insulin receptor binding of insulin, IGF-1, a single chainIGF^(B16B17) derivative peptide dimer and a two chain IGF^(B16B17)derivative peptide dimer. FIG. 7C is a graph demonstrating the relativeactivity of insulin, IGF-1, and a two chain IGF^(B16B17) derivativepeptide dimer to induce insulin receptor phosphorylation.

FIG. 8A-8C shows the degradation of a prodrug form of an IGF^(B16B17)derivative peptide: (Aib-Pro on (pNH₂—F)¹⁹ ofIGF1A(Ala)^(6,7,11,20)amide. The dipeptide was incubated in PBS, pH 7.4at 37° C. for predetermined lengths of time. Aliquots were taken at 20minutes (FIG. 8A), 81 minutes (FIG. 8B) and 120 minutes (FIG. 8C) afterbeginning the incubation, were quenched with 0.1% TFA and tested byanalytical HPLC. Peak a (IGF1A(Ala)^(6,7,11,20)(pNH₂—F)¹amide) and b(IGF1A(Ala)^(6,7,11,20)(Aib Pro-pNH—F)¹⁹amide) were identified withLC-MS and quantified by integration of peak area. The data indicate thespontaneous, non-enzymatic conversion ofIGF1A(Ala)^(6,7,11,20)(Aib-Pro-pNH—F)¹⁹amide toIGF1A(Ala)^(6,7,11,20)(pNH₂—F)¹amide over time.

FIGS. 9A & 9B are graphs depicting the in vitro activity of the prodrugAib,dPro-IGF1YL (dipeptide linked throughout the A19 4-aminoPhe). FIG.9A is a graph comparing relative insulin receptor binding of nativeinsulin (measured at 1 hour at 4° C.) and the A19 IGF prodrug analog(Aib,dPro-IGF1YL) over time (0 hours, 2.5 hours and 10.6 hours)incubated in PBS. FIG. 9B is a graph comparing relative insulin receptorbinding of native insulin (measured at 1.5 hour at 4° C.) and the A19IGF prodrug analog (Aib,dPro-IGF1YL) over time (0 hours, 1.5 hours and24.8 hours) incubated in 20% plasma/PBS. As indicated by the datapresented in the graph, increased activity is recovered form the A19 IGFprodrug analog sample as the prodrug form is converted to the activeIGF1YL peptide.

FIGS. 10A & 10B are graphs depicting the in vitro activity of theprodrug dK,(N-isobutylG)-IGF1YL (dipeptide linked throughout the A194-aminoPhe). FIG. 10A is a graph comparing relative insulin receptorbinding of native insulin (measured at 1 hour at 4° C.) and the A19 IGFprodrug analog (IGF1YL: dK,(N-isobutylG) over time (0 hours, 5 hours and52 hours) incubated in PBS. FIG. 10B is a graph comparing relativeinsulin receptor binding of native insulin (measured at 1.5 hour at 4°C.) and the A19 IGF prodrug analog (IGF1YL: dK,(N-isobutylG) over time(0 hours, 3.6 hours and 24.8 hours) incubated in 20% plasma/PBS. Asindicated by the data presented in the graph, increased activity isrecovered form the A19 IGF prodrug analog sample as the prodrug form isconverted to the active IGF1YL peptide.

FIGS. 11A & 11B are graphs depicting the in vitro activity of theprodrug dK(e-acetyl),Sar)-IGF1YL (dipeptide linked throughout the A194-aminoPhe). FIG. 11A is a graph comparing relative insulin receptorbinding of native insulin (measured at 1 hour at 4° C.) and the A19 IGFprodrug analog (IGF1YL: dK(e-acetyl),Sar) over time (0 hours, 7.2 hoursand 91.6 hours) incubated in PBS. FIG. 11B is a graph comparing relativeinsulin receptor binding of native insulin (measured at 1.5 hour at 4°C.) and the A19 IGF prodrug analog (IGF1YL: dK(e-acetyl),Sar) over time(0 hours, 9 hours and 95 hours) incubated in 20% plasma/PBS. Asindicated by the data presented in the graph, increased activity isrecovered from the A19 IGF prodrug analog sample as the prodrug form isconverted to the active IGF1YL peptide.

DETAILED DESCRIPTION Definitions

In describing and claiming the invention, the following terminology willbe used in accordance with the definitions set forth below.

As used herein, the term “prodrug” is defined as any compound thatundergoes chemical modification before exhibiting its pharmacologicaleffects.

A “bioactive polypeptide” refers to polypeptides which are capable ofexerting a biological effect in vitro and/or in vivo.

As used herein the term “amino acid” encompasses any molecule containingboth amino and carboxyl functional groups, wherein the amino andcarboxylate groups are attached to the same carbon (the alpha carbon).The alpha carbon optionally may have one or two further organicsubstituents. Designation of an amino acid without specifying itsstereochemistry is intended to encompass either the L or D form of theamino acid or a racemic mixture. However, in the instance where an aminoacid is designated by its three letter code and includes a superscriptnumber, the D form of the amino acid is specified by inclusion of alower case d before the three letter code and superscript number (e.g.,dLys⁻¹), wherein the designation lacking the lower case d (e.g., Lys⁻¹)is intended to specify the native L form of the amino acid. In thisnomenclature, the inclusion of the superscript number designates theposition of the amino acid in the IGF peptide sequence, wherein aminoacids that are located within the IGF sequence are designated bypositive superscript numbers numbered consecutively from the N-terminus.Additional amino acids linked to the IGF peptide either at theN-terminus or through a side chain are numbered starting with 0 andincreasing in negative integer value as they are further removed fromthe IGF sequence. For example, the position of an amino acid within adipeptide prodrug linked to the N-terminus of IGF is designatedaa⁻¹-aa⁰-IGF wherein aa⁰ represents the carboxy terminal amino acid ofthe dipeptide and aa⁻¹ designates the amino terminal amino acid of thedipeptide.

As used herein the term “hydroxyl acid” refers to amino acids that havebeen modified to replace the alpha carbon amino group with a hydroxylgroup.

As used herein the term “non-coded amino acid” encompasses any aminoacid that is not an L-isomer of any of the following 20 amino acids:Ala, Cys, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln,Arg, Ser, Thr, Val, Trp, Tyr.

A “dipeptide” is a compound formed by linkage of an alpha amino acid oran alpha hydroxyl acid to another amino acid, through a peptide bond.

As used herein the term “chemical cleavage” absent any furtherdesignation encompasses a non-enzymatic reaction that results in thebreakage of a covalent chemical bond.

A “bioactive polypeptide” refers to polypeptides which are capable ofexerting a biological effect in vitro and/or in vivo.

As used herein a general reference to a peptide is intended to encompasspeptides that have modified amino and carboxy termini. For example, anamino acid sequence designating the standard amino acids is intended toencompass standard amino acids at the N- and C-terminus as well as acorresponding hydroxyl acid at the N-terminus and/or a correspondingC-terminal amino acid modified to comprise an amide group in place ofthe terminal carboxylic acid.

As used herein an “acylated” amino acid is an amino acid comprising anacyl group which is non-native to a naturally-occurring amino acid,regardless by the means by which it is produced. Exemplary methods ofproducing acylated amino acids and acylated peptides are known in theart and include acylating an amino acid before inclusion in the peptideor peptide synthesis followed by chemical acylation of the peptide. Inone embodiment, the acyl group causes the peptide to have one or more of(i) a prolonged half-life in circulation, (ii) a delayed onset ofaction, (iii) an extended duration of action, (iv) an improvedresistance to proteases, such as DPP-IV, and (v) increased potency atthe insulin peptide receptor.

As used herein, an “alkylated” amino acid is an amino acid comprising analkyl group which is non-native to a naturally-occurring amino acid,regardless of the means by which it is produced. Exemplary methods ofproducing alkylated amino acids and alkylated peptides are known in theart and including alkylating an amino acid before inclusion in thepeptide or peptide synthesis followed by chemical alkylation of thepeptide. Without being held to any particular theory, it is believedthat alkylation of peptides will achieve similar, if not the same,effects as acylation of the peptides, e.g., a prolonged half-life incirculation, a delayed onset of action, an extended duration of action,an improved resistance to proteases, such as DPP-IV, and increasedpotency at the insulin peptide receptor.

As used herein, the term “pharmaceutically acceptable carrier” includesany of the standard pharmaceutical carriers, such as a phosphatebuffered saline solution, water, emulsions such as an oil/water orwater/oil emulsion, and various types of wetting agents. The term alsoencompasses any of the agents approved by a regulatory agency of the USFederal government or listed in the US Pharmacopeia for use in animals,including humans.

As used herein the term “pharmaceutically acceptable salt” refers tosalts of compounds that retain the biological activity of the parentcompound, and which are not biologically or otherwise undesirable. Manyof the compounds disclosed herein are capable of forming acid and/orbase salts by virtue of the presence of amino and/or carboxyl groups orgroups similar thereto.

Pharmaceutically acceptable base addition salts can be prepared frominorganic and organic bases. Salts derived from inorganic bases, includeby way of example only, sodium, potassium, lithium, ammonium, calciumand magnesium salts. Salts derived from organic bases include, but arenot limited to, salts of primary, secondary and tertiary amines.

Pharmaceutically acceptable acid addition salts may be prepared frominorganic and organic acids. Salts derived from inorganic acids includehydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,phosphoric acid, and the like. Salts derived from organic acids includeacetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid,malic acid, malonic acid, succinic acid, maleic acid, fumaric acid,tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid,methanesulfonic acid, ethanesulfonic acid, p-toluene-sulfonic acid,salicylic acid, and the like.

As used herein, the term “treating” includes prophylaxis of the specificdisorder or condition, or alleviation of the symptoms associated with aspecific disorder or condition and/or preventing or eliminating saidsymptoms. For example, as used herein the term “treating diabetes” willrefer in general to maintaining glucose blood levels near normal levelsand may include increasing or decreasing blood glucose levels dependingon a given situation.

As used herein an “effective” amount or a “therapeutically effectiveamount” of a prodrug refers to a nontoxic but sufficient amount of theprodrug to provide the desired effect. For example one desired effectwould be the prevention or treatment of hyperglycemia. The amount thatis “effective” will vary from subject to subject, depending on the ageand general condition of the individual, mode of administration, and thelike. Thus, it is not always possible to specify an exact “effectiveamount.” However, an appropriate “effective” amount in any individualcase may be determined by one of ordinary skill in the art using routineexperimentation.

The term, “parenteral” means not through the alimentary canal but bysome other route such as intranasal, inhalation, subcutaneous,intramuscular, intraspinal, or intravenous.

As used herein the term “native insulin peptide” is intended todesignate the 51 amino acid heterodimer comprising the A chain of SEQ IDNO: 1 and the B chain of SEQ ID NO: 2, as well as single-chain insulinanalogs that comprise SEQ ID NOS: 1 and 2. The term “insulin peptide” asused herein, absent further descriptive language is intended toencompass the 51 amino acid heterodimer comprising the A chain of SEQ IDNO: 1 and the B chain of SEQ ID NO: 2, as well as single-chain insulinanalogs thereof (including for example those disclosed in publishedinternational application WO96/34882 and U.S. Pat. No. 6,630,348, thedisclosures of which are incorporated herein by reference), and includesheterodimers and single-chain analogs that comprise modified derivativesof the native A chain and/or B chain, including modification of theamino acid at position A19, B16 or B25 to a 4-amino phenylalanine or oneor more amino acid substitutions at positions selected from A5, A8, A9,A10, A12, A14, A15, A17, A18, A21, B1, B2, B3, B4, B5, B9, B10, B13,B14, B17, B20, B21, B22, B23, B26, B27, B28, B29 and B30 or deletions ofany or all of positions B1-4 and B26-30. An “insulin prodrug analog” asused herein refers to an insulin peptide (or an IGF1-based insulinanalog as disclosed in Example 9) that has been modified by the covalentattachment of a dipeptide, via an amide linkage, at a location thatinterferes with insulin's or IGF1-based insulin analog's activity (e.g.,the ability to interact with the insulin and IGF-1 receptors).

As used herein, the term “single-chain insulin analog” encompasses agroup of structurally-related proteins wherein the insulin A and Bchains are covalently linked.

As used herein an amino acid “modification” refers to a substitution,addition or deletion of an amino acid, or the derivation of an aminoacid by the addition and/or removal of chemical groups to/from the aminoacid, and includes substitution with or addition of any of the 20 aminoacids commonly found in human proteins, as well as atypical ornon-naturally occurring amino acids. Commercial sources of atypicalamino acids include Sigma-Aldrich (Milwaukee, Wis.), ChemPep Inc.(Miami, Fla.), and Genzyme Pharmaceuticals (Cambridge, Mass.). Atypicalamino acids may be purchased from commercial suppliers, synthesized denovo, or chemically modified or derivatized from naturally occurringamino acids.

As used herein an amino acid “substitution” refers to the replacement ofone amino acid residue by a different amino acid residue. Throughout theapplication, all references to a particular amino acid position byletter and number (e.g. position A5) refer to the amino acid at thatposition of either the A chain (e.g. position A5) or the B chain (e.g.position B5) in the respective native human insulin A chain (SEQ IDNO: 1) or B chain (SEQ ID NO: 2), or the corresponding amino acidposition in any analogs thereof. For example, a reference herein to“position B28” absent any further elaboration would mean thecorresponding position B27 of the B chain of an insulin analog in whichthe first amino acid of SEQ ID NO: 2 has been deleted.

As used herein, the term “conservative amino acid substitution” isdefined herein as exchanges within one of the following five groups:

I. Small aliphatic, nonpolar or slightly polar residues:

-   -   Ala, Ser, Thr, Pro, Gly;

II. Polar, negatively charged residues and their amides:

-   -   Asp, Asn, Glu, Gln;

III. Polar, positively charged residues:

-   -   His, Arg, Lys; Ornithine (Orn)

IV. Large, aliphatic, nonpolar residues:

-   -   Met, Leu, Ile, Val, Cys, Norleucine (Nle), homocysteine

V. Large, aromatic residues:

-   -   Phe, Tyr, Trp, acetyl phenylalanine

As used herein the general term “polyethylene glycol chain” or “PEGchain”, refers to mixtures of condensation polymers of ethylene oxideand water, in a branched or straight chain, represented by the generalformula H(OCH₂CH₂)_(n)OH, wherein n is at least 9. Absent any furthercharacterization, the term is intended to include polymers of ethyleneglycol with an average total molecular weight selected from the range of500 to 80,000 Daltons. “Polyethylene glycol chain” or “PEG chain” isused in combination with a numeric suffix to indicate the approximateaverage molecular weight thereof. For example, PEG-5,000 refers topolyethylene glycol chain having a total molecular weight average ofabout 5,000 Daltons.

As used herein the term “pegylated” and like terms refers to a compoundthat has been modified from its native state by linking a polyethyleneglycol chain to the compound. A “pegylated polypeptide” is a polypeptidethat has a PEG chain covalently bound to the polypeptide.

As used herein a “linker” is a bond, molecule or group of molecules thatbinds two separate entities to one another. Linkers may provide foroptimal spacing of the two entities or may further supply a labilelinkage that allows the two entities to be separated from each other.Labile linkages include photocleavable groups, acid-labile moieties,base-labile moieties and enzyme-cleavable groups.

As used herein an “insulin dimer” is a complex comprising two insulinpeptides covalently bound to one another via a linker. The term insulindimer, when used absent any qualifying language, encompasses bothinsulin homodimers and insulin heterodimers. An insulin homodimercomprises two identical subunits (each comprising an A and B chain),whereas an insulin heterodimer comprises two subunits that differ,although the two subunits are substantially similar to one another.

The term “C₁-C_(n) alkyl” wherein n can be from 1 through 6, as usedherein, represents a branched or linear alkyl group having from one tothe specified number of carbon atoms. Typical C₁-C₆ alkyl groupsinclude, but are not limited to, methyl, ethyl, n-propyl, iso-propyl,butyl, iso-butyl, sec-butyl, tert-butyl, pentyl, hexyl and the like.

The terms “C₂-C_(n) alkenyl” wherein n can be from 2 through 6, as usedherein, represents an olefinically unsaturated branched or linear grouphaving from 2 to the specified number of carbon atoms and at least onedouble bond. Examples of such groups include, but are not limited to,1-propenyl, 2-propenyl (—CH₂—CH═CH₂), 1,3-butadienyl, (—CH═CHCH═CH₂),1-butenyl (—CH═CHCH₂CH₃), hexenyl, pentenyl, and the like.

The term “C₂-C_(n) alkynyl” wherein n can be from 2 to 6, refers to anunsaturated branched or linear group having from 2 to n carbon atoms andat least one triple bond. Examples of such groups include, but are notlimited to, 1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl, 1-pentynyl,and the like.

As used herein the term “aryl” refers to a mono- or bicyclic carbocyclicring system having one or two aromatic rings including, but not limitedto, phenyl, naphthyl, tetrahydronaphthyl, indanyl, indenyl, and thelike. The size of the aryl ring and the presence of substituents orlinking groups are indicated by designating the number of carbonspresent. For example, the term “(C₁-C₃ alkyl)(C₆-C₁₀ aryl)” refers to a5 to 10 membered aryl that is attached to a parent moiety via a one tothree membered alkyl chain.

The term “heteroaryl” as used herein refers to a mono- or bi-cyclic ringsystem containing one or two aromatic rings and containing at least onenitrogen, oxygen, or sulfur atom in an aromatic ring. The size of theheteroaryl ring and the presence of substituents or linking groups areindicated by designating the number of carbons present. For example, theterm “(C₁-C_(n) alkyl)(C₅-C₆heteroaryl)” refers to a 5 or 6 memberedheteroaryl that is attached to a parent moiety via a one to “n” memberedalkyl chain.

The term “C₃-C_(n) cycloalkyl” refers to a non-aromatic, monocyclic orpolycyclic ring comprising carbon and hydrogen atoms with the subscriptnumber indicating the number of carbon atoms present. For example theterm C₃-C₈ cycloalkyl represents the compounds cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl.

The term “C₃-C_(n) heterocyclic” refers to a cycloalkyl ring systemcontaining from one to “n−1” heteroatoms wherein the heteroatoms areselected from the group consisting of oxygen, sulfur, and nitrogen. Forexample the phrase “5-membered heterocycle” or “C₅ heterocycle”includes, but is not limited to, 5-membered heterocycles having onehetero atom (e.g. thiophenes, pyrroles, furans); 5-membered heterocycleshaving two heteroatoms in 1, 2 or 1,3 positions (e.g. oxazoles,pyrazoles, imidazoles, thiazoles, purines); 5-membered heterocycleshaving three heteroatoms (e.g. triazoles, thiadiazoles).

The term “C₃-C_(n) membered ring” as used herein refers to a saturatedor unsaturated hydrocarbon ring structure comprising a total of three to“n” number of elements linked to one another to form a ring, wherein thering elements are selected from the group consisting of C, O, S and N.The term is intended to encompass cycloalkyls, heterocyles, aryls andheteroaryls.

As used herein, the term “halo” refers to one or more members of thegroup consisting of fluorine, chlorine, bromine, and iodine.

As used herein the term “patient” without further designation isintended to encompass any warm blooded vertebrate domesticated animal(including for example, but not limited to livestock, horses, cats, dogsand other pets) and humans.

Embodiments

The present disclosure provides insulin prodrug derivatives that areformulated to delay onset of action and enhance the half life of theinsulin peptide, thus improving the therapeutic index of the underlyinginsulin peptide. The insulin prodrug chemistry disclosed herein allowsfor activation of the prodrug via a non-enzymatic degradation mechanism.The disclosed prodrug chemistry can be chemically conjugated to activesite amines to form amides that revert to the parent amine upondiketopiperazine formation and release of the prodrug element. Thisnovel biologically friendly prodrug chemistry spontaneously degradesunder physiological conditions (e.g. pH of about 7, at 37° C. in anaqueous environment) and is not reliant on enzymatic degradation. Theduration of the prodrug derivative is determined by the selection of thedipeptide prodrug sequence, and thus allows for flexibility in prodrugformulation.

In one embodiment a prodrug is provided having a non-enzymaticactivation half time (t½) of between 1-100 hrs under physiologicalconditions. Physiological conditions as disclosed herein are intended toinclude a temperature of about 35 to 40° C. and a pH of about 7.0 toabout 7.4 and more typically include a pH of 7.2 to 7.4 and atemperature of 36 to 38° C. in an aqueous environment. In one embodimenta dipeptide, capable of undergoing diketopiperazine formation underphysiological conditions, is covalently linked through an amide linkageto the insulin peptide.

Advantageously, the rate of cleavage, and thus activation of theprodrug, depends on the structure and stereochemistry of the dipeptidepro-moiety and also on the strength of the nucleophile. The prodrugsdisclosed herein will ultimately be chemically converted to structuresthat can be recognized by the native receptor of the drug, wherein thespeed of this chemical conversion will determine the time of onset andduration of in vivo biological action. The prodrug chemistry disclosedin this application relies upon an intramolecular chemical reaction thatis not dependent upon additional chemical additives, or enzymes. Thespeed of conversion is controlled by the chemical nature of thedipeptide substituent and its cleavage under physiological conditions.Since physiological pH and temperature are tightly regulated within ahighly defined range, the speed of conversion from prodrug to drug willexhibit high intra and interpatient reproducibility.

As disclosed herein prodrugs are provided wherein the bioactivepolypeptides have extended half lives of at least 1 hour, and moretypically greater than 20 hours but less than 100 hours, and areconverted to the active form at physiological conditions through anon-enzymatic reaction driven by inherent chemical instability. In oneembodiment the a non-enzymatic activation t½ time of the prodrug isbetween 1-100 hrs, and more typically between 12 and 72 hours, and inone embodiment the t½ is between 24-48 hrs as measured by incubating theprodrug in a phosphate buffer solution (e.g., PBS) at 37° C. and pH of7.2. The half lives of the various prodrugs are calculated by using theformula t_(1/2)=0.693/k, where ‘k’ is the first order rate constant forthe degradation of the prodrug. In one embodiment, activation of theprodrug occurs after cleavage of an amide bond linked dipeptide, andformation of a diketopiperazine or diketomorpholine, and the activeinsulin peptide.

Specific dipeptides composed of natural or synthetic amino acids havebeen identified that facilitate intramolecular decomposition underphysiological conditions to release active insulin peptides. Thedipeptide can be linked (via an amide bond) to an amino group present onnative insulin, or an amino group introduced into the insulin peptide bymodification of the native insulin peptide. In one embodiment thedipeptide structure is selected to resist cleavage by peptidases presentin mammalian sera, including for example dipeptidyl peptidase IV(DPP-IV). Accordingly, in one embodiment the rate of cleavage of thedipeptide prodrug element from the bioactive peptide is notsubstantially enhanced (e.g., greater than 2×) when the reaction isconducted using physiological conditions in the presence of serumproteases, relative to conducting the reaction in the absence of theproteases. Thus the cleavage half-life of the dipeptide prodrug elementfrom the insulin peptide (in PBS under physiological conditions) is notmore than two, three, four or five fold the cleavage half-life of thedipeptide prodrug element from the insulin peptide in a solutioncomprising a DPP-IV protease. In one embodiment the solution comprisinga DPP-IV protease is serum, more particularly mammalian serum, includinghuman serum.

In accordance with one embodiment the dipeptide prodrug elementcomprises the structure U-O, wherein U is an amino acid or a hydroxylacid and O is an N-alkylated amino acid. In one embodiment U, O, or theamino acid of the insulin peptide to which U-O is linked is a non-codedamino acid. In one embodiment U and/or O is an amino acid in the Dstereoisomer configuration. In some exemplary embodiments, U is an aminoacid in the D stereoisomer configuration and O is an amino acid in the Lstereoisomer configuration. In some exemplary embodiments, U is an aminoacid in the L stereoisomer configuration and O is an amino acid in the Dstereoisomer configuration. In some exemplary embodiments, U is an aminoacid in the D stereoisomer configuration and O is an amino acid in the Dstereoisomer configuration. In one embodiment O is an N-alkylated aminoacid but is not proline. In one embodiment the N-alkylated group ofamino acid O is a C₁-C₁₈ alkyl, and in one embodiment N-alkylated groupis C₁-C₆ alkyl. In one embodiment U-O is a dipeptide comprising thestructure of Formula I as defined herein.

In one embodiment the dipeptide is linked to the insulin peptide at anamino group selected from the N-terminal amino group of the A or Bchain, or the side chain amino group of an amino acid present at anactive site of the insulin peptide. In accordance with one embodimentthe dipeptide extension is covalently linked to an insulin peptidethrough the side chain amine of a lysine residue that resides at or nearthe active site. In one embodiment the dipeptide extension is attachedthrough a synthetic amino acid or a modified amino acid, wherein thesynthetic amino acid or modified amino acid exhibits a functional groupsuitable for covalent attachment of the dipeptide extension (e.g., thearomatic amine of amino-phenylalanine). In accordance with oneembodiment the dipeptide is linked to the insulin peptide at an aminogroup selected from the N-terminal amino group of the A or B chain, orthe side chain amino group of an aromatic amine (e.g., a4-amino-phenylalanine residue) present at position A19, B16 or B25. Inone embodiment the U-O dipeptide is bound at position A19 through a4-amino phenylalanine present at position A19.

The dipeptide prodrug element is designed to spontaneously cleave itsamide linkage to the insulin analog under physiological conditions andin the absence of enzymatic activity. In one embodiment the N-terminalamino acid of the dipeptide extension comprises a C-alkylated amino acid(e.g. amino isobutyric acid). In one embodiment the C-terminal aminoacid of the dipeptide comprises an N-alkylated amino acid (e.g., prolineor N-methyl glycine). In one embodiment the dipeptide comprises thesequence of an N-terminal C-alkylated amino acid followed by anN-alkylated amino acid.

Applicants have discovered that the selective insertion of a 4-aminophenyl amino acid moiety for the native tyrosine at position 19 of the Achain can be accommodated without loss in potency of the insulin peptide(see FIG. 3). Subsequent chemical amidation of this active site aminogroup with the dipeptide prodrug moiety disclosed herein dramaticallylessens insulin receptor binding activity and thus provides a suitableprodrug of insulin (see FIG. 6, data provided for the IGF1Y¹⁶L¹⁷(p-NH₂—F)^(A19) analog which has been demonstrated to have comparableactivity as insulin (p-NH₂—F)^(A19), see FIG. 4). Accordingly, in oneembodiment the dipeptide prodrug element is linked to the aromatic ringof an A19 4-aminophenylalanine via an amide bond, wherein the C-terminalamino acid of the dipeptide comprises an N-alkylated amino acid and theN-terminal amino acid of the dipeptide is any amino acid.

The dipeptide prodrug moiety can also be attached to additional sites ofan insulin peptide to prepare a prodrug or depot analog of insulin. Inaccordance with one embodiment an insulin prodrug/depot analog isprovided comprising an A chain, a B chain, and a dipeptide linked via anamide bond to one or more sites selected from the group consisting ofthe N-terminal amino group of the A chain or B chain, or the side chainamino group of an internal amino acid including for example, linked tothe aromatic amine of a 4-amino-phenylalanine residue present atposition A19, B16 or B25. In one embodiment the insulin peptidecomprises two dipeptide elements, wherein the dipeptide elements areoptionally pegylated, alkylated, acylated or linked to a depot polymer.In one embodiment the dipeptide comprises an N-terminal C-alkylatedamino acid followed by an N-alkylated amino acid.

The A chain and B chain comprising the insulin prodrug analog maycomprise the native sequence of the respective peptides (i.e., SEQ IDNO: 1 and SEQ ID NO: 2) or may comprise a derivative of SEQ ID NO: 1and/or SEQ ID NO: 2 wherein the derivative includes modification of theamino acid at position A19, B16 or B25 to a 4-amino phenylalanine and/orone or more amino acid substitutions at positions selected from A5, A8,A9, A10, A14, A15, A17, A18, A19 and A21, B1, B2, B3, B4, B5, B9, B10,B13, B14, B17, B20, B22, B23, B26, B27, B28, B29 and B30 or deletions ofany or all of positions B1-4 and B26-30. In one embodiment the dipeptideprodrug element is linked to an N-terminal amino group of the A or Bchain, wherein the C-terminal amino acid of the dipeptide prodrugelement comprises an N-alkylated amino acid and the N-terminal aminoacid of the dipeptide prodrug element is any amino acid, with theproviso that when the C-terminal amino acid of the dipeptide is proline,the N-terminal amino acid of the dipeptide comprises a C-alkylated aminoacid.

In one embodiment the dipeptide prodrug element comprises the generalstructure of Formula I:

wherein

R₁, R₂, R₄ and R₈ are independently selected from the group consistingof H, C₁-C₁₈ alkyl, C₂-C₁₈ alkenyl, (C₁-C₁₈ alkyl)OH, (C₁-C₁₈ alkyl)SH,(C₂-C₃ alkyl)SCH₃, (C₁-C₄ alkyl)CONH₂, (C₁-C₄ alkyl)COOH, (C₁-C₄alkyl)NH₂, (C₁-C₄ alkyl)NHC(NH₂ ⁺)NH₂, (C₀-C₄ alkyl)(C₃-C₆ cycloalkyl),(C₀-C₄ alkyl)(C₂-C₅ heterocyclic), (C₀-C₄ alkyl)(C₆-C₁₀ aryl)R₇, (C₁-C₄alkyl)(C₃-C₉ heteroaryl), and C₁-C₁₂ alkyl(W)C₁-C₁₂ alkyl, wherein W isa heteroatom selected from the group consisting of N, S and O, or R₁ andR₂ together with the atoms to which they are attached form a C₃-C₁₂cycloalkyl or aryl; or R₄ and R₈ together with the atoms to which theyare attached form a C₃-C₆ cycloalkyl;

R₃ is selected from the group consisting of C₁-C₁₈ alkyl, (C₁-C₁₈alkyl)OH, (C₁-C₁₈ alkyl)NH₂, (C₁-C₁₈ alkyl)SH, (C₀-C₄alkyl)(C₃-C₆)cycloalkyl, (C₀-C₄ alkyl)(C₂-C₅ heterocyclic), (C₀-C₄alkyl)(C₆-C₁₀ aryl)R₇, and (C₁-C₄ alkyl)(C₃-C₉ heteroaryl) or R₄ and R₃together with the atoms to which they are attached form a 4, 5 or 6member heterocyclic ring;

R₅ is NHR₆ or OH;

R₆ is H, C₁-C₈ alkyl or R₆ and R₂ together with the atoms to which theyare attached form a 4, 5 or 6 member heterocyclic ring; and

R₇ is selected from the group consisting of H and OH, with the provisothat when R₄ and R₃ together with the atoms to which they are attachedform a 4, 5 or 6 member heterocyclic ring, both R₁ and R₂ are other thanH.

In another embodiment the dipeptide prodrug element comprises thegeneral structure:

wherein

R₁ and R₈ are independently H or C₁-C₈ alkyl;

R₂ and R₄ are independently selected from the group consisting of H,C₁-C₈ alkyl, C₂-C₈ alkenyl, (C₁-C₄ alkyl)OH, (C₁-C₄ alkyl)SH, (C₂-C₃alkyl)SCH₃, (C₁-C₄ alkyl)CONH₂, (C₁-C₄ alkyl)COOH, (C₁-C₄ alkyl)NH₂,(C₁-C₄ alkyl)NHC(NH₂+) NH₂, (C₀-C₄ alkyl)(C₃-C₆ cycloalkyl), (C₀-C₄alkyl)(C₂-C₅ heterocyclic), (C₀-C₄ alkyl)(C₆-C₁₀ aryl)R₇, and CH₂(C₃-C₉heteroaryl), or R₁ and R₂ together with the atoms to which they areattached form a C₃-C₁₂ cycloalkyl;

R₃ is selected from the group consisting of C₁-C₈ alkyl, (C₁-C₄alkyl)OH, (C₁-C₄ alkyl)NH₂, (C₁-C₄ alkyl)SH, (C₃-C₆)cycloalkyl or R₄ andR₃ together with the atoms to which they are attached form a 5 or 6member heterocyclic ring;

R₅ is NHR₆ or OH;

R₆ is H, C₁-C₈ alkyl, or R₆ and R₂ together with the atoms to which theyare attached form a 5 or 6 member heterocyclic ring; and

R₇ is selected from the group consisting of hydrogen, C₁-C₁₈ alkyl,C₂-C₁₈ alkenyl, (C₀-C₄ alkyl)CONH₂, (C₀-C₄ alkyl)COOH, (C₀-C₄ alkyl)NH₂,(C₀-C₄ alkyl)OH and halo, provided that when the dipeptide of Formula Iis linked through the N-terminal amine of a peptide and R₄ and R₃together with the atoms to which they are attached form a 5 or 6 memberheterocyclic ring, both R₁ and R₂ are not H. In one embodiment eitherthe first amino acid and/or the second amino acid of the dipeptideprodrug element is an amino acid in the D stereoisomer configuration.

In one embodiment the prodrug element of Formula I is provided whereinR₁ is selected from the group consisting of H and C₁-C₈ alkyl; and

-   -   R₂ and R₄ are independently selected from the group consisting        of H, C₁-C₈ alkyl, C₂-C₈ alkenyl, (C₁-C₄ alkyl)OH, (C₁-C₄        alkyl)SH, (C₂-C₃ alkyl)SCH₃, (C₁-C₄ alkyl)CONH₂, (C₁-C₄        alkyl)COOH, (C₁-C₄ alkyl)NH₂, (C₁-C₄ alkyl)NHC(NH₂ ⁺) NH₂,        (C₀-C₄ alkyl)(C₃-C₆ cycloalkyl), (C₀-C₄ alkyl)(C₆-C₁₀ aryl)R₇,        and CH₂(C₅-C₉ heteroaryl), or R₁ and R₂ together with the atoms        to which they are attached form a C₃-C₈ cycloalkyl ring;    -   R₃ is selected from the group consisting of C₁-C₈ alkyl, (C₁-C₄        alkyl)OH, (C₁-C₄ alkyl)SH, (C₁-C₄ alkyl)NH₂, (C₃-C₆)cycloalkyl        or R₄ and R₃ together with the atoms to which they are attached        form a 5 or 6 member heterocyclic ring;    -   R₅ is NHR₆ or OH;    -   R₆ is H, or R₆ and R₂ together with the atoms to which they are        attached form a 5 or 6 member heterocyclic ring;    -   R₇ is selected from the group consisting of H and OH and R₈        is H. In one embodiment R₃ is C₁-C₈ alkyl and R₄ is selected        from the group consisting of H, C₁-C₆ alkyl, CH₂OH, (C₀-C₄        alkyl)(C₆-C₁₀ aryl)R₇, and CH₂(C₅-C₉ heteroaryl) or R₄ and R₃        together with the atoms to which they are attached form a 5 or 6        member heterocyclic ring. In a further embodiment R₅ is NHR₆ and        R₈ is H.

In a further embodiment the prodrug element of Formula I is providedwherein

-   -   R₁ is selected from the group consisting of H and C₁-C₈ alkyl;        and    -   R₂ and R₄ are independently selected from the group consisting        of H, C₁-C₈ alkyl, C₂-C₈ alkenyl, (C₁-C₄ alkyl)OH, (C₁-C₄        alkyl)SH, (C₂-C₃ alkyl)SCH₃, (C₁-C₄ alkyl)CONH₂, (C₁-C₄        alkyl)COOH, (C₁-C₄ alkyl)NH₂, (C₁-C₄ alkyl)NHC(NH₂ ⁺) NH₂,        (C₀-C₄ alkyl)(C₃-C₆ cycloalkyl), (C₀-C₄ alkyl)(C₆-C₁₀ aryl)R₇,        and CH₂(C₅-C₉ heteroaryl), or R₁ and R₂ together with the atoms        to which they are attached form a C₃-C₈ cycloalkyl ring;    -   R₃ is selected from the group consisting of C₁-C₈ alkyl, (C₁-C₄        alkyl)OH, (C₁-C₄ alkyl)SH, (C₁-C₄ alkyl)NH₂, (C₃-C₆)cycloalkyl        or R₄ and R₃ together with the atoms to which they are attached        form a 5 or 6 member heterocyclic ring;    -   R₅ is NHR₆ or OH;    -   R₆ is H, or R₆ and R₂ together with the atoms to which they are        attached form a 5 or 6 member heterocyclic ring;    -   R₇ is selected from the group consisting of hydrogen, C₁-C₁₈        alkyl, C₂-C₁₈ alkenyl, (C₀-C₄ alkyl)CONH₂, (C₀-C₄ alkyl)COOH,        (C₀-C₄ alkyl)NH₂, (C₀-C₄ alkyl)OH and halo; and R₈ is H,        provided that when R₄ and R₃ together with the atoms to which        they are attached form a 5 or 6 member heterocyclic ring, both        R₁ and R₂ are not H. In one embodiment either the first amino        acid and/or the second amino acid of the dipeptide prodrug        element is a non-coded amino acid and in one embodiment is an        amino acid in the D stereoisomer configuration.

In other embodiments the dipeptide prodrug element has the structure ofFormula I, wherein

R₁ and R₈ are independently H or C₁-C₈ alkyl;

R₂ and R₄ are independently selected from the group consisting of H,C₁-C₈ alkyl, C₂-C₈ alkenyl, (C₁-C₄ alkyl)OH, (C₁-C₄ alkyl)SH, (C₂-C₃alkyl)SCH₃, (C₁-C₄ alkyl)CONH₂, (C₁-C₄ alkyl)COOH, (C₁-C₄ alkyl)NH₂,(C₁-C₄ alkyl)NHC(NH₂+) NH₂, (C₀-C₄ alkyl)(C₃-C₆ cycloalkyl), (C₀-C₄alkyl)(C₂-C₅ heterocyclic), (C₀-C₄ alkyl)(C₆-C₁₀ aryl)R₇, and CH₂(C₃-C₉heteroaryl), or R₁ and R₂ together with the atoms to which they areattached form a C₃-C₁₂ cycloalkyl;

R₃ is C₁-C₁₈ alkyl;

R₅ is NHR₆;

R₆ is H or C₁-C₈ alkyl;

R₇ is selected from the group consisting of hydrogen, C₁-C₁₈ alkyl,C₂-C₁₈ alkenyl, (C₀-C₄ alkyl)CONH₂, (C₀-C₄ alkyl)COOH, (C₀-C₄ alkyl)NH₂,(C₀-C₄ alkyl)OH, and halo and R₈ is H.

In a further embodiment the dipeptide prodrug element has the structureof Formula I, wherein

R₁ and R₂ are independently C₁-C₁₈ alkyl or (C₀-C₄ alkyl)(C₆-C₁₀aryl)R₇; or R₁ and R₂ are linked through —(CH₂)_(p), wherein p is 2-9;

R₃ is C₁-C₁₈ alkyl;

R₄ and R₈ are each hydrogen;

R₅ is NH₂; and

R₇ is selected from the group consisting of hydrogen, C₁-C₁₈ alkyl,C₂-C₁₈ alkenyl, (C₀-C₄ alkyl)CONH₂, (C₀-C₄ alkyl)COOH, (C₀-C₄ alkyl)NH₂,(C₀-C₄ alkyl)OH, and halo.

In a further embodiment the dipeptide prodrug element has the structureof Formula I, wherein

R₁ and R₂ are independently selected from the group consisting ofhydrogen, C₁-C₁₈ alkyl, (C₁-C₁₈ alkyl)OH, (C₁-C₄ alkyl)NH₂, and (C₀-C₄alkyl)(C₆-C₁₀ aryl)R₇, or R₁ and R₂ are linked through (CH₂)_(p),wherein p is 2-9;

R₃ is C₁-C₁₈ alkyl or R₃ and R₄ together with the atoms to which theyare attached form a 4-12 heterocyclic ring;

R₄ and R₈ are independently selected from the group consisting ofhydrogen, C₁-C₈ alkyl and (C₀-C₄ alkyl)(C₆-C₁₀ aryl)R₇;

R₅ is NH₂; and

R₇ is selected from the group consisting of H, C₁-C₁₈ alkyl, C₂-C₁₈alkenyl, (C₀-C₄ alkyl)CONH₂, (C₀-C₄ alkyl)COOH, (C₀-C₄ alkyl)NH₂, (C₀-C₄alkyl)OH, and halo, with the proviso that both R₁ and R₂ are nothydrogen and provided that at least one of R₄ or R₈ is hydrogen.

In another embodiment the dipeptide prodrug element has the structure ofFormula I, wherein

R₁ and R₂ are independently selected from the group consisting ofhydrogen, C₁-C₈ alkyl and (C₁-C₄ alkyl)NH₂, or R₁ and R₂ are linkedthrough (CH₂)_(p), wherein p is 2-9;

R₃ is C₁-C₈ alkyl or R₃ and R₄ together with the atoms to which they areattached form a 4-6 heterocyclic ring;

R₄ is selected from the group consisting of hydrogen and C₁-C₈ alkyl;

R₈ is hydrogen; and

R₅ is NH₂, with the proviso that both R₁ and R₂ are not hydrogen.

In a further embodiment the dipeptide prodrug element has the structureof Formula I, wherein

R₁ and R₂ are independently selected from the group consisting ofhydrogen, C₁-C₈ alkyl and (C₁-C₄ alkyl)NH₂;

R₃ is C₁-C₆ alkyl;

R₄ and R₈ are each hydrogen; and

R₅ is NH₂, with the proviso that both R₁ and R₂ are not hydrogen.

In another embodiment the dipeptide prodrug element has the structure ofFormula I, wherein

R₁ and R₂ are independently selected from the group consisting ofhydrogen and C₁-C₈ alkyl, (C₁-C₄ alkyl)NH₂, or R₁ and R₂ are linkedthrough (CH₂)_(p), wherein p is 2-9;

R₃ is C₁-C₈ alkyl;

R₄ is (C₀-C₄ alkyl)(C₆-C₁₀ aryl)R₇;

R₅ is NH₂;

R₇ is selected from the group consisting of hydrogen, C₁-C₈ alkyl and(C₀-C₄ alkyl)OH; and

R₈ is hydrogen, with the proviso that both R₁ and R₂ are not hydrogen.

In another embodiment the dipeptide prodrug element has the structure ofFormula I, wherein

R₁ is selected from the group consisting of hydrogen, C₁-C₈ alkyl and(C₀-C₄ alkyl)(C₆-C₁₀ aryl)R₇;

R₂ is hydrogen;

R₃ is C₁-C₁₈ alkyl;

R₄ and R₈ are each hydrogen;

R₅ is NHR₆ or OH;

R₆ is H, C₁-C₈ alkyl, or R₆ and R₁ together with the atoms to which theyare attached form a 4, 5 or 6 member heterocyclic ring; and

R₇ is selected from the group consisting of hydrogen, C₁-C₁₈ alkyl,C₂-C₁₈ alkenyl, (C₀-C₄ alkyl)CONH₂, (C₀-C₄ alkyl)COOH, (C₀-C₄ alkyl)NH₂,(C₀-C₄ alkyl)OH, and halo, with the proviso that, if R₁ is alkyl or(C₀-C₄ alkyl)(C₆-C₁₀ aryl)R₇, then R₁ and R₅ together with the atoms towhich they are attached form a 4-11 heterocyclic ring.

In accordance with one embodiment an insulin prodrug analog is providedcomprising an insulin peptide and an amide linked dipeptide. Moreparticularly, the insulin prodrug analog comprises an A chain sequenceand a B chain sequence wherein the A chain comprises the sequenceZ-GIVEQCCX₁SICSLYQLENX₂CX₃-R₁₃ (SEQ ID NO: 3), or an analog thereofcomprising a sequence that differs from SEQ ID NO: 3 by 1 to 9, 1 to 5or 1 to 3 amino acid modifications, selected from positions A5, A8, A9,A10, A14, A15, A17, A18 (relative to the native insulin A chain), andthe B chain sequence comprises the sequence ofJ-X₁₄-X₄LCGX₅X₆LVEALX₇LVCGERGFX₈ (SEQ ID NO: 14), or an analog thereofcomprising a sequence that differs from SEQ ID NO: 14 sequence by 1 to10, 1 to 5 or 1 to 3 amino acid modifications, selected from positionsB1, B2, B3, B4, B5, B13, B14, B17, B20, B22, B23, B26, B27, B28, B29 andB30 (relative to the native insulin B chain; i.e., amino acid X₄ of SEQID NO: 14 corresponds to position B5 in native insulin). Z and J areindependently H or a dipeptide comprising the general structure ofFormula I:

X₁₄ is either a bond joining the “J” element to the X₄LCGX₅X₆LVEALX₇LVCGERGFX₈ (SEQ ID NO: 14) sequence or X₁₄ represents a 1 to 4 amino acidsequence selected from the group consisting of a X₉VNQ (SEQ ID NO: 21),VNQ, NQ and Q that joins the “J” element to the X₄LCGX₅X₆LVEALX₇LVCGERGFX₈ (SEQ ID NO: 14) sequence.

X₁ is selected from the group consisting of threonine and histidine;

X₂ is an amino acid of the general structure

-   -   wherein m is an integer selected from 0-3 and X is selected from        the group consisting of OH, NH₂, NHR₁₀ and OCH₃, wherein R₁₀ is        H or a dipeptide comprising the general structure of Formula I:

X₃ is selected from the group consisting of asparagine, glycine,alanine, threonine and serine;

X₄ is selected from the group consisting of histidine and threonine;

X₅ is selected from the group consisting of alanine, glycine and serine;

X₆ is selected from the group consisting of histidine, aspartic acid,glutamic acid, homocysteic acid and cysteic acid;

X₇ is an amino acid of the general structure

-   -   wherein m is an integer selected from 0-3 and X₁₂ is selected        from the group consisting of OH, NH₂, NHR₁₁ and OCH₃, wherein        R₁₁ is H or a dipeptide comprising the general structure of        Formula I:

X₈ is an amino acid of the general structure

-   -   wherein m is an integer selected from 0-3 and X₁₃ is selected        from the group consisting of H, OH, NH₂, NHR₁₂ and OCH₃, wherein        R₁₂ is H or a dipeptide comprising the general structure of        Formula I:

X₉ is selected from the group consisting of phenylalanine anddesamino-phenylalanine; wherein

R₁, R₂, R₄ and R₈ are independently selected from the group consistingof H, C₁-C₁₈ alkyl, C₂-C₁₈ alkenyl, (C₁-C₁₈ alkyl)OH, (C₁-C₁₈ alkyl)SH,(C₂-C₃ alkyl)SCH₃, (C₁-C₄ alkyl)CONH₂, (C₁-C₄ alkyl)COOH, (C₁-C₄alkyl)NH₂, (C₁-C₄ alkyl)NHC(NH₂ ⁺)NH₂, (C₀-C₄ alkyl)(C₃-C₆ cycloalkyl),(C₀-C₄ alkyl)(C₂-C₅ heterocyclic), (C₀-C₄ alkyl)(C₆-C₁₀ aryl)R₇, (C₁-C₄alkyl)(C₃-C₉ heteroaryl), and C₁-C₁₂ alkyl(W)C₁-C₁₂ alkyl, wherein W isa heteroatom selected from the group consisting of N, S and O, or R₁ andR₂ together with the atoms to which they are attached form a C₃-C₁₂cycloalkyl or aryl; or R₄ and R₈ together with the atoms to which theyare attached form a C₃-C₆ cycloalkyl;

R₃ is selected from the group consisting of C₁-C₁₈ alkyl, (C₁-C₁₈alkyl)OH, (C₁-C₁₈ alkyl)NH₂, (C₁-C₁₈ alkyl)SH, (C₀-C₄alkyl)(C₃-C₆)cycloalkyl, (C₀-C₄ alkyl)(C₂-C₅ heterocyclic), (C₀-C₄alkyl)(C₆-C₁₀ aryl)R₇, and (C₁-C₄ alkyl)(C₃-C₉ heteroaryl) or R₄ and R₃together with the atoms to which they are attached form a 4, 5 or 6member heterocyclic ring;

R₅ is NHR₆ or OH;

R₆ is H, C₁-C₈ alkyl or R₆ and R₂ together with the atoms to which theyare attached form a 4, 5 or 6 member heterocyclic ring; and

R₇ is selected from the group consisting of H and OH; and

R₁₃ is COOH or CONH₂. In one embodiment R₁₃ is COOH and the carboxyterminal amino acid of the B chain has an amide (CONH₂) in place of thenatural alpha carbon carboxy group. In one embodiment one or more of X,X₁₂, X₁₃, J and Z is a dipeptide comprising the general structure ofFormula I:

and in one embodiment two of X, X₁₂, X₁₃, J and Z comprise a dipeptideof the general structure of Formula I:

In accordance with one embodiment at least one of X, X₁₂, X₁₃, J and Zis a dipeptide comprising the general structure of Formula I:

and in one embodiment one and only one of X, X₁₂, X₁₃, J and Z comprisesa dipeptide of the general structure of Formula I:

(i.e., only one dipeptide prodrug element is attached to the insulinpeptide). Furthermore, when the dipeptide prodrug element is linked tothe N-terminus of the A or B chain (i.e., either J or Z comprise thedipeptide) and R₄ and R₃ together with the atoms to which they areattached form a 4, 5 or 6 member heterocyclic ring, then at least one ofR₁ and R₂ are other than H, and in one embodiment both R₁ and R₂ areother than H. In one embodiment J and Z are both H, X₁₂ is OH, X₁₃ is Hor OH, and X is NHR₁₀ wherein R₁₀ a dipeptide comprising the generalstructure of Formula I. In a further embodiment, the A chain comprisesthe sequence GIVEQCCX₁SICSLYQLENX₂CX₃-R₁₃ (SEQ ID NO: 3), the B chainsequence comprises the sequence of X₁₄-X₄LCGX₅X₆LVEALX₇LVCGERGFX₈ (SEQID NO: 14), m is 1, X₁₂ is OH, X₁₃ is H or OH, X is NHR₁₀, wherein R₁₀ adipeptide comprising the general structure of Formula I, R₁₃ is COOH andthe carboxy terminal amino acid of the B chain has an amide (CONH₂) inplace of the natural alpha carbon carboxy group, with the remainingdesignations defined as immediately above.

In one embodiment the dipeptide present at X, X₁₂, X₁₃, J and Z is adipeptide comprising the general structure of Formula I:

wherein

R₁, R₂, R₄ and R₈ are independently selected from the group consistingof H, C₁-C₁₈ alkyl, C₂-C₁₈ alkenyl, (C₁-C₁₈ alkyl)OH, (C₁-C₁₈ alkyl)SH,(C₂-C₃ alkyl)SCH₃, (C₁-C₄ alkyl)CONH₂, (C₁-C₄ alkyl)COOH, (C₁-C₄alkyl)NH₂, (C₁-C₄ alkyl)NHC(NH₂ ⁺)NH₂, (C₀-C₄ alkyl)(C₃-C₆ cycloalkyl),(C₀-C₄ alkyl)(C₂-C₅ heterocyclic), (C₀-C₄ alkyl)(C₆-C₁₀ aryl)R₇, (C₁-C₄alkyl)(C₃-C₉ heteroaryl), and C₁-C₁₂ alkyl(W₁)C₁-C₁₂ alkyl, wherein W₁is a heteroatom selected from the group consisting of N, S and O, or R₁and R₂ together with the atoms to which they are attached form a C₃-C₁₂cycloalkyl; or R₄ and R₈ together with the atoms to which they areattached form a C₃-C₆ cycloalkyl;

R₃ is selected from the group consisting of C₁-C₁₈ alkyl, (C₁-C₁₈alkyl)OH, (C₁-C₁₈ alkyl)NH₂, (C₁-C₁₈ alkyl)SH, (C₀-C₄alkyl)(C₃-C₆)cycloalkyl, (C₀-C₄ alkyl)(C₂-C₅ heterocyclic), (C₀-C₄alkyl)(C₆-C₁₀ aryl)R₇, and (C₁-C₄ alkyl)(C₃-C₉ heteroaryl) or R₄ and R₃together with the atoms to which they are attached form a 4, 5 or 6member heterocyclic ring;

R₅ is NHR₆ or OH;

R₆ is H, C₁-C₈ alkyl or R₆ and R₁ together with the atoms to which theyare attached form a 4, 5 or 6 member heterocyclic ring; and

R₇ is selected from the group consisting of hydrogen, C₁-C₁₈ alkyl,C₂-C₁₈ alkenyl, (C₀-C₄ alkyl)CONH₂, (C₀-C₄ alkyl)COOH, (C₀-C₄ alkyl)NH₂,(C₀-C₄ alkyl)OH, and halo provided that when R₄ and R₃ together with theatoms to which they are attached form a 5 or 6 member heterocyclic ring,both R₁ and R₂ are not H. In one embodiment when J or Z comprise thedipeptide of Formula I, and R₄ and R₃ together with the atoms to whichthey are attached form a 4, 5 or 6 member heterocyclic ring, then bothR₁ and R₂ are not hydrogen.

In one embodiment R₁ is selected from the group consisting of H andC₁-C₈ alkyl;

-   -   R₂ and R₄ are independently selected from the group consisting        of H, C₁-C₈ alkyl, C₂-C₈ alkenyl, (C₁-C₄ alkyl)OH, (C₁-C₄        alkyl)SH, (C₂-C₃ alkyl)SCH₃, (C₁-C₄ alkyl)CONH₂, (C₁-C₄        alkyl)COOH, (C₁-C₄ alkyl)NH₂, (C₁-C₄ alkyl)NHC(NH₂ ⁺) NH₂,        (C₀-C₄ alkyl)(C₃-C₆ cycloalkyl), (C₀-C₄ alkyl)(C₆-C₁₀ aryl)R₇,        and CH₂(C₅-C₉ heteroaryl);    -   R₃ is selected from the group consisting of C₁-C₈ alkyl, (C₁-C₄        alkyl)OH, (C₁-C₄ alkyl)SH, (C₁-C₄ alkyl)NH₂, (C₃-C₆)cycloalkyl        or R₄ and R₃ together with the atoms to which they are attached        form a 5 or 6 member heterocyclic ring;    -   R₅ is NHR₆ or OH;    -   R₆ is H, or R₆ and R₂ together with the atoms to which they are        attached form a 5 or 6 member heterocyclic ring;    -   R₇ is selected from the group consisting of H and OH and R₈        is H. In accordance with another embodiment, m is 1, R₈ is H, R₃        is C₁-C₆ alkyl and R₄ is selected from the group consisting of        H, C₁-C₄ alkyl, (C₃-C₆)cycloalkyl, (C₁-C₄ alkyl)OH, (C₁-C₄        alkyl)SH and (C₀-C₄ alkyl)(C₆ aryl)R₇, or R₃ and R₄ together        with the atoms to which they are attached form a 5 member        heterocyclic ring. In one embodiment, m is 1, R₈ is H, R₃ is        C₁-C₆ alkyl and R₄ is selected from the group consisting of H,        C₁-C₄ alkyl or R₃ and R₄ together with the atoms to which they        are attached form a 5 member heterocyclic ring. In a further        embodiment X₉ is phenylalanine and a single dipeptide extension        is linked to the insulin peptide through an amide bond to the        N-terminus of the A chain or the B chain. In an alternative        embodiment the insulin peptide comprises a 4 amino phenylalanine        substitution at position 19 of the A chain and a single        dipeptide extension is linked to the insulin peptide through an        amide bond formed at the aromatic amine of the 4 amino        phenylalanine. In one embodiment insulin analogs disclosed        herein comprise a C-terminal amide or ester in place of a        C-terminal carboxylate on the A chain and/or B chain.

In accordance with one embodiment the dipeptide of Formula I is furthermodified to comprise a large polymer that interferes with the insulinanalog's ability to interact with the insulin or IGF-1 receptor.Subsequent cleavage of the dipeptide releases the insulin analog fromthe dipeptide complex wherein the released insulin analog is fullyactive. In accordance with one embodiment J comprises the dipeptide ofFormula I, wherein the dipeptide of Formula I is further modified tocomprises a large polymer that interferes with the bound insulinanalog's ability to interact with the insulin or IGF-1 receptor. Inaccordance with one embodiment one of X, X₁₂, X₁₃, J and Z comprises adipeptide of the general structure of Formula I:

wherein the dipeptide of Formula I is pegylated, alkylated or acylated.In one embodiment either J, Z or X comprises an acylated or pegylateddipeptide of Formula I, and in one embodiment J comprises an acylated orpegylated dipeptide of Formula I.

In one embodiment, the dipeptide prodrug element is covalently bound tothe insulin peptide via an amide linkage at the N-terminus of the Achain or the B chain, or attached to an amine bearing side chain of aninternal amino acid, wherein the dipeptide further comprises a depotpolymer linked to dipeptide. In one embodiment a native amino acid ofthe insulin peptide is substituted with an amino acid suitable forforming an amide bond with the dipeptide of Formula I. In one embodimenta depot bearing dipeptide is linked at a position selected from A14,A19, B16, B28 and B29. In one embodiment two or more depot polymers arelinked to a single dipeptide element. The depot polymer is selected tobe biocompatible and of sufficient size that the insulin peptidemodified by covalent attachment of the dipeptide remains sequestered atan injection site and/or incapable of interacting with its correspondingreceptor upon administration to a patient. Subsequent cleavage of thedipeptide releases the insulin peptide to interact with its intendedtarget.

In accordance with one embodiment the depot polymer is selected frombiocompatible polymers known to those skilled in the art. The depotpolymers typically have a size selected from a range of about 20,000 to120,000 Daltons. In one embodiment the depot polymer has a size selectedfrom a range of about 40,000 to 100,000 or about 40,000 to 80,000Daltons. In one embodiment the depot polymer has a size of about 40,000,50,000, 60,000, 70,000 or 80,000 Daltons. Suitable depot polymersinclude but are not limited to dextrans, polylactides, polyglycolides,caprolactone-based polymers, poly(caprolactone), polyanhydrides,polyamines, polyesteramides, polyorthoesters, polydioxanones,polyacetals, polyketals, polycarbonates, polyphosphoesters, polyesters,polybutylene terephthalate, polyorthocarbonates, polyphosphazenes,succinates, poly(malic acid), poly(amino acids), polyvinylpyrrolidone,polyethylene glycol, polyhydroxycellulose, polysaccharides, chitin,chitosan, hyaluronic acid, and copolymers, terpolymers and mixturesthereof, and biodegradable polymers and their copolymers includingcaprolactone-based polymers, polycaprolactones and copolymers whichinclude polybutylene terephthalate. In one embodiment the depot polymeris selected from the group consisting of polyethylene glycol, dextran,polylactic acid, polyglycolic acid and a copolymer of lactic acid andglycolic acid, and in one specific embodiment the depot polymer ispolyethylene glycol. In one embodiment the depot polymer is polyethyleneglycol and the combined molecular weight of depot polymer(s) linked tothe dipeptide element is about 40,000 to 80,000 Daltons.

In accordance with one embodiment the dipeptide of Formula I furthercomprises a polyethylene oxide, alkyl or acyl group. In one embodimentone or more polyethylene oxide chains are linked to the dipeptide ofFormula I wherein the combined molecular weight of the polyethyleneoxide chains ranges from about 20,000 to about 80,000 Daltons, or 40,000to 80,000 Daltons or 40,000 to 60,000 Daltons. In one embodiment thepolyethylene oxide is polyethylene glycol. In one embodiment at leastone polyethylene glycol chain having a molecular weight of about 40,000Daltons is linked to the dipeptide of Formula I. In another embodimentthe dipeptide of Formula I is acylated with an acyl group of sufficientsize to bind serum albumin, and thus inactivate the insulin analog uponadministration. The acyl group can be linear or branched, and in oneembodiment is a C16 to C30 fatty acid. For example, the acyl group canbe any of a C16 fatty acid, C18 fatty acid, C20 fatty acid, C22 fattyacid, C24 fatty acid, C26 fatty acid, C28 fatty acid, or a C30 fattyacid. In one embodiment, the acyl group is a C16 to C20 fatty acid,e.g., a C18 fatty acid or a C20 fatty acid.

In accordance with one embodiment an insulin prodrug analog is providedcomprising an A chain sequence and a B chain sequence wherein the Achain comprises the sequence GIVEQCCX₁SICSLYQLENX₂CX₃-R₁₃ (SEQ ID NO:3), and the B chain sequence comprises the sequence ofX₁₄-X₄LCGX₅X₆LVEALX₇LVCGERGFX₈ (SEQ ID NO: 14), wherein

X₁₄ is selected from the group consisting of an N-terminal amine, X₉VNQ(SEQ ID NO: 21), VNQ, NQ and Q that joins the “J” element to theX₄LCGX₅X₆LVEALX₇LVCG ERGFX₈-R₁₄ (SEQ ID NO: 14) sequence.

X₁ is selected from the group consisting of threonine and histidine;

X₂ is an amino acid of the general structure

wherein U is an amino acid or a hydroxyl acid and O is an N-alkylatedamino acid linked through an amide bond;

X₃ is selected from the group consisting of asparagine, ornithine,glycine, alanine, threonine and serine;

X₄ is selected from the group consisting of histidine and threonine;

X₅ is selected from the group consisting of alanine, glycine and serine;

X₆ is selected from the group consisting of histidine, aspartic acid,glutamic acid, homocysteic acid and cysteic acid;

X₇ is an amino acid of the general structure

-   -   wherein m is an integer selected from 0-3 and X₁₂ is selected        from the group consisting of OH, NH₂ and OCH₃;

X₈ is an amino acid of the general structure

-   -   wherein m is an integer selected from 0-3 and X₁₃ is selected        from the group consisting of H, OH, NH₂ and OCH₃;

X₉ is selected from the group consisting of phenylalanine anddesamino-phenylalanine; and R₁₃ and R₁₄ are independently COOH or CONH₂.In one embodiment, X₇ and X₈ are both tyrosine, R₁₃ is COOH and thecarboxy terminal amino acid of the B chain has an amide (CONH₂) in placeof the natural alpha carbon carboxy group.

In accordance with one embodiment a compound comprising the sequenceZ-GIVEQCCX₁SICSLYQLENX₂CX₃ (SEQ ID NO: 3), or an analog thereofcomprising a sequence that differs from SEQ ID NO: 3 by 1 to 3 aminoacid modifications, selected from positions A5, A8, A9, A10, A14, A15,A17, A18 (relative to the native insulin A chain sequence) is provided.In this embodiment X₁ is selected from the group consisting of threonineand histidine;

X₂ is an amino acid of the general structure

-   -   wherein X is selected from the group consisting of OH, NH₂,        NHR₁₀ and OCH₃,

further wherein R₁₀ and Z are independently H or a dipeptide of thestructure U-O, wherein U is an amino acid or a hydroxyl acid and O is anN-alkylated amino acid, wherein O is linked to the peptide of SEQ ID NO:3 through formation of an amide bond, with the proviso that R₁₀ and Zare not the same and that U, O or the amino acid of SEQ ID NO: 3 towhich U-O is linked is a non-coded amino acid. In one embodiment thechemical cleavage of the dipeptide from SEQ ID NO: 3 is at least about90% complete within about 1 to about 720 hours in PBS underphysiological conditions. In another embodiment the chemical cleavagehalf-life (t_(1/2)) of U-O from SEQ ID NO: 3 is at least about 1 hour toabout 1 week in PBS under physiological conditions. The compound in oneembodiment further comprises an insulin B chain linked to the A chain ofSEQ ID NO: 3 either through intermolecular disulfide linkages or as arecombinant single chain polypeptide.

Selection of the substituents on the dipeptide element and theattachment site of the dipeptide prodrug element can impact the rate ofchemical cleavage of the dipeptide prodrug element from the insulinpeptide. In one embodiment an insulin prodrug is provided comprising anA chain sequence and a B chain sequence, wherein the A chain comprisesthe sequence GIVEQCCX₁SICSLYQLENX₂CX₃ (SEQ ID NO: 3) and the B chainsequence comprises the sequence of X₁₄-X₄LCGX₅X₆LVEALYLVCGERGFF (SEQ IDNO: 4) wherein

X₁ is selected from the group consisting of threonine and histidine;

X₂ is an amino acid of the general structure

-   -   wherein X is selected from the group consisting of OH, NH₂, and        OCH₃;

X₃ is selected from the group consisting of asparagine, glycine,alanine, threonine and serine;

X₄ is selected from the group consisting of histidine and threonine;

X₅ is selected from the group consisting of alanine, glycine and serine;

X₆ is selected from the group consisting of histidine, aspartic acid,glutamic acid, homocysteic acid and cysteic acid; and

X₁₄ is selected from the group consisting of a bond, X₉VNQ (SEQ ID NO:21), VNQ, NQ and Q, and X₉ is selected from the group consisting ofphenylalanine and desamino-phenylalanine, further wherein a dipeptideprodrug element comprises the structure:

is linked to the alpha amino group of the N-terminal amino acid of thepeptide of SEQ ID NO: 3 or SEQ ID NO: 4 with the proviso that when R₄and R₃ together with the atoms to which they are attached form a 4, 5 or6 member heterocyclic ring, then both R₁ and R₂ are not hydrogen. Inthis embodiment, compounds having a t_(1/2) of about 1 hour in PBS underphysiological conditions are provided wherein

R₁ and R₂ are independently C₁-C₁₈ alkyl or aryl; or R₁ and R₂ arelinked through —(CH₂)_(p)—, wherein p is 2-9;

R₃ is C₁-C₁₈ alkyl;

R₄ and R₈ are each hydrogen; and

R₅ is an amine.

In other embodiments, prodrugs having the prodrug element linked at theN-terminus and having a t_(1/2) of, e.g., about 1 hour comprise adipeptide prodrug element with the structure:

wherein

R₁ and R₂ are independently C₁-C₁₈ alkyl or (C₀-C₄ alkyl)(C₆-C₁₀aryl)R₇; or R₁ and R₂ are linked through —(CH₂)_(p), wherein p is 2-9;

R₃ is C₁-C₁₈ alkyl;

R₄ and R₈ are each hydrogen;

R₅ is NH₂; and

R₇ is selected from the group consisting of hydrogen, C₁-C₁₈ alkyl,C₂-C₁₈ alkenyl, (C₀-C₄ alkyl)CONH₂, (C₀-C₄ alkyl)COOH, (C₀-C₄ alkyl)NH₂,(C₀-C₄ alkyl)OH, and halo.

Alternatively, an insulin prodrug is provided comprising an A chainsequence of GIVEQCCX₁SICSLYQLENX₂CX₃ (SEQ ID NO: 3) and a B chainsequence of X₁₄-X₄LCGX₅X₆LVEALX₇LVCGERGFX₈ (SEQ ID NO: 14) wherein thedipeptide prodrug element is linked to the alpha amino group of theN-terminal amino acid of the peptide of SEQ ID NO: 3 or SEQ ID NO: 14and exhibits a t_(1/2) between about 6 to about 24 hours in PBS underphysiological conditions. In one embodiment such compounds comprise aprodrug element of Formula I, wherein

R₁ and R₂ are independently selected from the group consisting ofhydrogen, C₁-C₁₈ alkyl and aryl, or R₁ and R₂ are linked through—(CH₂)_(p)—, wherein p is 2-9;

R₃ is C₁-C₁₈ alkyl or R₃ and R₄ together with the atoms to which theyare attached form a 4-12 heterocyclic ring;

R₄ and R₈ are independently selected from the group consisting ofhydrogen, C₁-C₈ alkyl and aryl; and

R₅ is an amine, with the proviso that both R₁ and R₂ are not hydrogenand provided that one of R₄ or R₈ is hydrogen.

In a further embodiment an insulin prodrug is provided comprising an Achain sequence of GIVEQCCX₁SICSLYQLENX₂CX₃ (SEQ ID NO: 3) and a B chainsequence of X₁₄-X₄LCGX₅X₆LVEALX₇LVCGERGFX₈ (SEQ ID NO: 14) wherein thedipeptide prodrug element is linked to the alpha amino group of theN-terminal amino acid of the peptide of SEQ ID NO: 3 or SEQ ID NO: 14and exhibits a t_(1/2) of about 72 to about 168 hours in PBS underphysiological conditions. In one embodiment such compounds comprise aprodrug element of Formula I, wherein

R₁ is selected from the group consisting of hydrogen, C₁-C₈ alkyl andaryl;

R₂ is H;

R₃ is C₁-C₁₈ alkyl;

R₄ and R₈ are each hydrogen; and

R₅ is an amine or N-substituted amine or a hydroxyl;

with the proviso that, if R₁ is alkyl or aryl, then R₁ and R₅ togetherwith the atoms to which they are attached form a 4-11 heterocyclic ring.

In one embodiment, prodrugs having the dipeptide prodrug element linkedto the N-terminal alpha amino acid of the insulin A or B chain peptideand having a t_(1/2), e.g., between about 12 to about 72 hours, or inone embodiment between about 12 to about 48 hours, comprise a dipeptideprodrug element with the structure:

wherein R₁ and R₂ are independently selected from the group consistingof hydrogen, C₁-C₁₈ alkyl, (C₁-C₁₈ alkyl)OH, (C₁-C₄ alkyl)NH₂, and(C₀-C₄ alkyl)(C₆-C₁₀ aryl)R₇, or R₁ and R₂ are linked through (CH₂)_(p),wherein p is 2-9;

R₃ is C₁-C₁₈ alkyl or R₃ and R₄ together with the atoms to which theyare attached form a 4-12 heterocyclic ring;

R₄ and R₈ are independently selected from the group consisting ofhydrogen, C₁-C₈ alkyl and (C₀-C₄ alkyl)(C₆-C₁₀ aryl)R₇;

R₅ is NH₂; and

R₇ is selected from the group consisting of H, C₁-C₁₈ alkyl, C₂-C₁₈alkenyl, (C₀-C₄ alkyl)CONH₂, (C₀-C₄ alkyl)COOH, (C₀-C₄ alkyl)NH₂, (C₀-C₄alkyl)OH, and halo; with the proviso that both R₁ and R₂ are nothydrogen and provided that at least one of R₄ or R₈ is hydrogen.

In one embodiment, prodrugs having the dipeptide prodrug element linkedto the N-terminal amino acid of the insulin A or B chain peptide andhaving a t_(1/2), e.g., between about 12 to about 72 hours, or in oneembodiment between about 12 to about 48 hours, comprise a dipeptideprodrug element with the structure:

wherein R₁ and R₂ are independently selected from the group consistingof hydrogen, C₁-C₈ alkyl and (C₁-C₄ alkyl)NH₂, or R₁ and R₂ are linkedthrough (CH₂)_(p), wherein p is 2-9;

R₃ is C₁-C₈ alkyl or R₃ and R₄ together with the atoms to which they areattached form a 4-6 heterocyclic ring;

R₄ is selected from the group consisting of hydrogen and C₁-C₈ alkyl;and

R₅ is NH₂. with the proviso that both R₁ and R₂ are not hydrogen.

In other embodiments, prodrugs having the dipeptide prodrug elementlinked to the N-terminal amino acid of the insulin A or B chain peptideand having a t_(1/2), e.g., between about 12 to about 72 hours, or inone embodiment between about 12 to about 48 hours, comprise a dipeptideprodrug element with the structure:

wherein

R₁ and R₂ are independently selected from the group consisting ofhydrogen, C₁-C₈ alkyl and (C₁-C₄ alkyl)NH₂;

R₃ is C₁-C₆ alkyl;

R₄ is hydrogen; and

R₅ is NH₂. with the proviso that both R₁ and R₂ are not hydrogen.

In one embodiment, prodrugs having the dipeptide prodrug element linkedto the N-terminal amino acid of the insulin A or B chain peptide andhaving a t_(1/2), e.g., between about 12 to about 72 hours, or in oneembodiment between about 12 to about 48 hours, comprise a dipeptideprodrug element with the structure:

wherein

R₁ and R₂ are independently selected from the group consisting ofhydrogen and C₁-C₈ alkyl, (C₁-C₄ alkyl)NH₂, or R₁ and R₂ are linkedthrough (CH₂)_(p), wherein p is 2-9;

R₃ is C₁-C₈ alkyl;

R₄ is (C₀-C₄ alkyl)(C₆-C₁₀ aryl)R₇;

R₅ is NH₂; and

R₇ is selected from the group consisting of hydrogen, C₁-C₈ alkyl and(C₀-C₄ alkyl)OH. with the proviso that both R₁ and R₂ are not hydrogen.

In addition a prodrug having the dipeptide prodrug element linked to theN-terminal alpha amino acid of the insulin A or B chain peptide andhaving a t_(1/2), e.g., of about 72 to about 168 hours is providedwherein the dipeptide prodrug element has the structure:

wherein R₁ is selected from the group consisting of hydrogen, C₁-C₈alkyl and (C₀-C₄ alkyl)(C₆-C₁₀ aryl)R₇;

R₃ is C₁-C₁₈ alkyl;

R₄ and R₈ are each hydrogen;

R₅ is NHR₆ or OH;

R₆ is H, C₁-C₈ alkyl, or R₆ and R₁ together with the atoms to which theyare attached form a 4, 5 or 6 member heterocyclic ring; and

R₇ is selected from the group consisting of hydrogen, C₁-C₁₈ alkyl,C₂-C₁₈ alkenyl, (C₀-C₄ alkyl)CONH₂, (C₀-C₄ alkyl)COOH, (C₀-C₄ alkyl)NH₂,(C₀-C₄ alkyl)OH, and halo. with the proviso that, if R₁ is alkyl or(C₀-C₄ alkyl)(C₆-C₁₀ aryl)R₇, then R₁ and R₅ together with the atoms towhich they are attached form a 4-11 heterocyclic ring.

In one embodiment the dipeptide prodrug element is linked to a sidechain amine of an internal amino acid of the insulin peptide. In thisembodiment prodrugs having a t_(1/2), e.g., of about 1 hour have thestructure:

wherein

R₁ and R₂ are independently C₁-C₈ alkyl or (C₀-C₄ alkyl)(C₆-C₁₀ aryl)R₇;or R₁ and R₂ are linked through —(CH₂)_(p)—, wherein p is 2-9;

R₃ is C₁-C₁₈ alkyl;

R₄ and R₈ are each hydrogen;

R₅ is NH₂; and

R₇ is selected from the group consisting of hydrogen, C₁-C₁₈ alkyl,C₂-C₁₈ alkenyl, (C₀-C₄ alkyl)CONH₂, (C₀-C₄ alkyl)COOH, (C₀-C₄ alkyl)NH₂,(C₀-C₄ alkyl)OH, and halo.

Furthermore, prodrugs having a t_(1/2), e.g., between about 6 to about24 hours and having the dipeptide prodrug element linked to an internalamino acid side chain comprise a dipeptide prodrug element with thestructure:

wherein R₁ and R₂ are independently selected from the group consistingof hydrogen, C₁-C₈ alkyl, and (C₀-C₄ alkyl)(C₆-C₁₀ aryl)R₇, or R₁ and R₂are linked through —(CH₂)_(p)—, wherein p is 2-9;

R₃ is C₁-C₁₈ alkyl or R₃ and R₄ together with the atoms to which theyare attached form a 4-12 heterocyclic ring;

R₄ and R₈ are independently hydrogen, C₁-C₁₈ alkyl or (C₀-C₄alkyl)(C₆-C₁₀ aryl)R₇;

R₅ is NHR₆;

R₆ is H or C₁-C₈ alkyl, or R₆ and R₂ together with the atoms to whichthey are attached form a 4, 5 or 6 member heterocyclic ring; and

R₇ is selected from the group consisting of hydrogen, C₁-C₁₈ alkyl,C₂-C₁₈ alkenyl, (C₀-C₄ alkyl)CONH₂, (C₀-C₄ alkyl)COOH, (C₀-C₄ alkyl)NH₂,(C₀-C₄ alkyl)OH, and halo, with the proviso that both R₁ and R₂ are nothydrogen and provided that at least one of R₄ or R₈ is hydrogen.

In addition a prodrug having a t_(1/2), e.g., of about 72 to about 168hours and having the dipeptide prodrug element linked to an internalamino acid side chain of the insulin peptide is provided wherein thedipeptide prodrug element has the structure:

wherein R₁ is selected from the group consisting of hydrogen, C₁-C₁₈alkyl and (C₀-C₄ alkyl)(C₆-C₁₀ aryl)R₇;

R₃ is C₁-C₁₈ alkyl;

R₄ and R₈ are each hydrogen;

R₅ is NHR₆ or OH;

R₆ is H or C₁-C₈ alkyl, or R₆ and R₁ together with the atoms to whichthey are attached form a 4, 5 or 6 member heterocyclic ring; and

R₇ is selected from the group consisting of hydrogen, C₁-C₁₈ alkyl,C₂-C₁₈ alkenyl, (C₀-C₄ alkyl)CONH₂, (C₀-C₄ alkyl)COOH, (C₀-C₄ alkyl)NH₂,(C₀-C₄ alkyl)OH, and halo; with the proviso that, if R₁ and R₂ are bothindependently an alkyl or (C₀-C₄ alkyl)(C₆-C₁₀ aryl)R₇, either R₁ or R₂is linked through (CH₂)_(p) to R₅, wherein p is 2-9.

In one embodiment the dipeptide prodrug element is linked to a sidechain amine of an internal amino acid of the insulin peptide wherein theinternal amino acid comprises the structure of Formula IV:

wherein

n is an integer selected from 1 to 4. In one embodiment n is 3 or 4 andin one embodiment the internal amino acid is lysine. In one embodimentthe dipeptide prodrug element is linked to a primary amine on a sidechain of an amino acid located at position 28, or 29 of the B-chain ofthe insulin peptide.

In one embodiment an insulin prodrug is provided comprising an A chainsequence and a B chain sequence, wherein the A chain comprises thesequence GIVEQCCX₁SICSLYQLENX₂CX₃ (SEQ ID NO: 3) and the B chainsequence comprises the sequence of X₁₄-X₄LCGX₅X₆LVEALX₇LVCGERGFX₈ (SEQID NO: 14) wherein

X₁ is selected from the group consisting of threonine and histidine;

X₂ is an amino acid of the general structure

-   -   wherein X is selected from the group consisting of OH, NH₂,        NHR₁₀ and OCH₃, wherein R₁₀ is H or a dipeptide comprising the        general structure:

wherein

-   -   R₁ and R₂ are independently selected from the group consisting        of hydrogen, C₁-C₈ alkyl, and (C₀-C₄ alkyl)(C₆-C₁₀ aryl)R₇, or        R₁ and R₂ are linked through —(CH₂)_(p)—, wherein p is 2-9;    -   R₃ is C₁-C₁₈ alkyl or R₃ and R₄ together with the atoms to which        they are attached form a 4-12 heterocyclic ring;    -   R₄ and R₈ are independently hydrogen, C₁-C₁₈ alkyl or (C₀-C₄        alkyl)(C₆-C₁₀ aryl)R₇;    -   R₅ is NHR₆;    -   R₆ is H or C₁-C₈ alkyl, or R₆ and R₂ together with the atoms to        which they are attached form a 4, 5 or 6 member heterocyclic        ring; and    -   R₇ is selected from the group consisting of hydrogen, C₁-C₁₈        alkyl, C₂-C₁₈ alkenyl, (C₀-C₄ alkyl)CONH₂, (C₀-C₄ alkyl)COOH,        (C₀-C₄ alkyl)NH₂, (C₀-C₄ alkyl)OH, and halo;

X₃ is selected from the group consisting of asparagine, glycine,alanine, threonine and serine;

X₄ is selected from the group consisting of histidine and threonine;

X₅ is selected from the group consisting of alanine, glycine and serine;

X₆ is selected from the group consisting of histidine, aspartic acid,glutamic acid, homocysteic acid and cysteic acid;

X₇ is an amino acid of the general structure

-   -   wherein X₁₂ is selected from the group consisting of OH, NH₂,        NHR₁₁ and OCH₃, wherein R₁₁ is H or a dipeptide comprising the        general structure:

X₈ is an amino acid of the general structure

-   -   wherein X₁₃ is selected from the group consisting of H, OH, NH₂,        NHR₁₂ and OCH₃, wherein R₁₂ is H or a dipeptide comprising the        general structure:

X₁₄ is selected from the group consisting of a Hydrogen (forming anN-terminal amine), X₉VNQ (SEQ ID NO: 21), VNQ, NQ and Q, and X₉ isselected from the group consisting of phenylalanine anddesamino-phenylalanine, with the proviso that one and only one of R₁₀,R₁₁ and R₁₂ is a dipeptide comprising the general structure:

In accordance with one embodiment wherein the dipeptide prodrug elementis linked to an amino substituent of an aryl group of an aromatic aminoacid, prodrug formulations can be provided having the desired time ofactivation. For example, an insulin prodrug comprising the structure ofFormula III:

wherein m is an integer from 0 to 3 and having a t½ of about 1 hour inPBS under physiological conditions is provided. In one embodiment wherean insulin prodrug comprises the structure of formula III and exhibitssuch a half life,

R₁ and R₂ are independently C₁-C₁₈ alkyl or aryl;

R₃ is C₁-C₁₈ alkyl or R₃ and R₄ together with the atoms to which theyare attached form a 4-12 heterocyclic ring;

R₄ and R₈ are independently selected from the group consisting ofhydrogen, C₁-C₁₈ alkyl and aryl; and

R₅ is an amine or a hydroxyl. In one embodiment m is 1.

In one embodiment, the dipeptide prodrug element is linked to theinsulin peptide via an amine present on an aryl group of an aromaticamino acid of the insulin peptide, wherein prodrugs having a t_(1/2),e.g., of about 1 hour have a dipeptide structure of:

wherein R₁ and R₂ are independently C₁-C₁₈ alkyl or (C₀-C₄ alkyl)(C₆-C₁₀aryl)R₇;

R₃ is C₁-C₁₈ alkyl or R₃ and R₄ together with the atoms to which theyare attached form a 4-12 heterocyclic ring;

R₄ and R₈ are independently selected from the group consisting ofhydrogen, C₁-C₁₈ alkyl and (C₀-C₄ alkyl)(C₆-C₁₀ aryl)R₇;

R₅ is NH₂ or OH; and

R₇ is selected from the group consisting of hydrogen, C₁-C₁₈ alkyl,C₂-C₁₈ alkenyl, (C₀-C₄ alkyl)CONH₂, (C₀-C₄ alkyl)COOH, (C₀-C₄ alkyl)NH₂,(C₀-C₄ alkyl)OH, and halo.

In another embodiment an insulin prodrug comprising the structure ofFormula III, wherein m is an integer from 0 to 3 and has a t½ of about 6to about 24 hours in PBS under physiological conditions, is provided. Inone embodiment where an insulin prodrug comprises the structure ofFormula III and exhibits such a half life,

R₁ and R₂ are independently selected from the group consisting ofhydrogen, C₁-C₁₈ alkyl and aryl, or R₁ and R₂ are linked through—(CH₂)_(p)—, wherein p is 2-9;

R₃ is C₁-C₁₈ alkyl or R₃ and R₄ together with the atoms to which theyare attached form a 4-6 heterocyclic ring;

R₄ and R₈ are independently selected from the group consisting ofhydrogen, C₁-C₁₈ alkyl and aryl; and

R₅ is an amine or N-substituted amine. In one embodiment m is 1.

In one embodiment, prodrugs having the dipeptide prodrug element linkedvia an amine present on an aryl group of an aromatic amino acid andhaving a t_(1/2), e.g., of about 6 to about 24 hours are providedwherein the dipeptide comprises a structure of:

wherein

R₁ is selected from the group consisting of hydrogen, C₁-C₁₈ alkyl,(C₁-C₁₈ alkyl)OH, (C₁-C₄ alkyl)NH₂, and (C₀-C₄ alkyl)(C₆-C₁₀ aryl)R₇;

R₃ is C₁-C₁₈ alkyl or R₃ and R₄ together with the atoms to which theyare attached form a 4-6 heterocyclic ring;

R₄ and R₈ are independently selected from the group consisting ofhydrogen, C₁-C₁₈ alkyl and (C₀-C₄ alkyl)(C₆-C₁₀ aryl)R₇;

R₅ is NHR₆;

R₆ is H, C₁-C₈ alkyl, or R₆ and R₁ together with the atoms to which theyare attached form a 4, 5 or 6 member heterocyclic ring; and

R₇ is selected from the group consisting of hydrogen, C₁-C₁₈ alkyl,C₂-C₁₈ alkenyl, (C₀-C₄ alkyl)CONH₂, (C₀-C₄ alkyl)COOH, (C₀-C₄ alkyl)NH₂,(C₀-C₄ alkyl)OH, and halo.

In another embodiment an insulin prodrug comprising the structure ofFormula III, wherein m is an integer from 0 to 3 and has a t½ of about72 to about 168 hours in PBS under physiological conditions, isprovided. In one embodiment where an insulin prodrug comprises thestructure of formula III and exhibits such a half life,

R₁ and R₂ are independently selected from the group consisting ofhydrogen, C₁-C₈ alkyl and aryl;

R₃ is C₁-C₁₈ alkyl or R₃ and R₄ together with the atoms to which theyare attached form a 4-6 heterocyclic ring;

R₄ and R₈ are each hydrogen; and

R₅ is selected from the group consisting of amine, N-substituted amineand hydroxyl. In one embodiment m is 1.

In one embodiment, prodrugs having the dipeptide prodrug element linkedvia an aromatic amino acid and having a t_(1/2), e.g., of about 72 toabout 168 hours are provided wherein the dipeptide comprises a structureof:

wherein R₁ is selected from the group consisting of hydrogen, C₁-C₈alkyl, (C₁-C₄ alkyl)COOH, and (C₀-C₄ alkyl)(C₆-C₁₀ aryl)R₇, or R₁ and R₅together with the atoms to which they are attached form a 4-11heterocyclic ring;

R₃ is C₁-C₁₈ alkyl or R₃ and R₄ together with the atoms to which theyare attached form a 4-6 heterocyclic ring;

R₄ is hydrogen or forms a 4-6 heterocyclic ring with R₃;

R₈ is hydrogen;

R₅ is NHR₆ or OH;

R₆ is H or C₁-C₈ alkyl, or R₆ and R₁ together with the atoms to whichthey are attached form a 4, 5 or 6 member heterocyclic ring; and

R₇ is selected from the group consisting of hydrogen, C₁-C₁₈ alkyl,C₂-C₁₈ alkenyl, (C₀-C₄ alkyl)CONH₂, (C₀-C₄ alkyl)COOH, (C₀-C₄ alkyl)NH₂,(C₀-C₄ alkyl)OH, and halo.

In one embodiment the insulin prodrug analog comprises an A chainsequence of GIVEQCCX₁SICSLYQLENX₂CX₃-R₁₃ (SEQ ID NO: 3) and a B chainsequence of X₁₄-X₄LCGX₅X₆LVEALX₇LVCGERGFX₈ (SEQ ID NO: 14) wherein

X₁ is selected from the group consisting of threonine, histidine,arginine and lysine;

X₂ is an amino acid of the general structure

-   -   wherein X is selected from the group consisting of OH, NH₂, and        OCH₃;

X₃ is asparagine, glycine, alanine, threonine, or serine.

X₄ is selected from the group consisting of histidine and threonine;

X₅ is selected from the group consisting of alanine, glycine and serine;

X₆ is selected from the group consisting of histidine, aspartic acid,glutamic acid, homocysteic acid and cysteic acid;

X₇ is tyrosine;

X₈ is tyrosine or phenylalanine;

X₉ is selected from the group consisting of phenylalanine anddesamino-phenylalanine;

X₁₀ is aspartate-lysine dipeptide, a lysine-proline dipeptide, or aproline-lysine dipeptide;

X₁₁ is threonine, alanine, or a threonine-arginine-arginine tripeptide;further wherein the B chain comprises a carboxy terminus extension of 1to 4 amino acids wherein said carboxy terminal extension comprises anamino acid having the structure of

-   -   wherein m is an integer from 0-3;    -   n is an integer from 1-4;    -   R₁₂ is a dipeptide comprising the general structure:

-   -   R₁ is selected from the group consisting of H and C₁-C₈ alkyl;        and    -   R₂ and R₄ are independently selected from the group consisting        of H, C₁-C₈ alkyl, C₂-C₈ alkenyl, (C₁-C₄ alkyl)OH, (C₁-C₄        alkyl)SH, (C₂-C₃ alkyl)SCH₃, (C₁-C₄ alkyl)CONH₂, (C₁-C₄        alkyl)COOH, (C₁-C₄ alkyl)NH₂, (C₁-C₄ alkyl)NHC(NH₂ ⁺) NH₂,        (C₀-C₄ alkyl)(C₃-C₆ cycloalkyl), (C₀-C₄ alkyl)(C₆-C₁₀ aryl)R₇,        and CH₂(C₅-C₉ heteroaryl);    -   R₃ is selected from the group consisting of C₁-C₈ alkyl, (C₁-C₄        alkyl)OH, (C₁-C₄ alkyl)SH, (C₁-C₄ alkyl)NH₂, (C₃-C₆)cycloalkyl        or R₄ and R₃ together with the atoms to which they are attached        form a 5 or 6 member heterocyclic ring;    -   R₅ is NHR₆ or OH;    -   R₆ is H, or R₆ and R₂ together with the atoms to which they are        attached form a 5 or 6 member heterocyclic ring;    -   R₇ is selected from the group consisting of hydrogen, C₁-C₁₈        alkyl, C₂-C₁₈ alkenyl, (C₀-C₄ alkyl)CONH₂, (C₀-C₄ alkyl)COOH,        (C₀-C₄ alkyl)NH₂, (C₀-C₄ alkyl)OH, and halo; and    -   R₁₃ is COOH or CONH₂,

In one embodiment the insulin prodrug analog comprises an A chainsequence that includes the sequence GIVEQCCX₁SICSLYQLENX₂CX₃-R₁₃ (SEQ IDNO: 3) and a B chain sequence that includes the sequence ofJ-X₁₄-X₄LCGX₅X₆LVEALX₇LVCGERGFX₈ (SEQ ID NO: 14) orJ-X₉VNQX₄LCGX₅X₆LVEALX₇LVCGERGFX₈YTX₁₀ X₁₁-R₁₄ (SEQ ID NO: 5) wherein Jis H (forming an N-terminal amine) or a dipeptide comprising the generalstructure:

wherein X₁₄ is either a bond joining the “J” element to SEQ ID NO: 14 orX₁₄ represents a 1 to 4 amino acid sequence selected from the groupconsisting of a FVNQ (SEQ ID NO: 11), VNQ, NQ and Q that joins the “J”element to SEQ ID NO: 14;

X₁ is selected from the group consisting of threonine, histidine,arginine and lysine;

X₂ is an amino acid of the general structure

-   -   wherein X is selected from the group consisting of OH, NH₂, and        OCH₃;

X₃ is asparagine, glycine, alanine, threonine, or serine.

X₄ is selected from the group consisting of histidine and threonine;

X₅ is selected from the group consisting of alanine, glycine and serine;

X₆ is selected from the group consisting of histidine, aspartic acid,glutamic acid, homocysteic acid and cysteic acid;

X₇ is an amino acid of the general structure

-   -   wherein X₁₂ is selected from the group consisting of OH, OCH₃,        NH₂ and NHR₁₁, wherein R₁₁ is a dipeptide comprising the general        structure:

X₈ is an amino acid of the general structure

-   -   wherein X₁₃ is selected from the group consisting of H, OH,        OCH₃, NH₂, and NHR₁₂, wherein R₁₂ is a dipeptide comprising the        general structure:

X₉ is selected from the group consisting of phenylalanine anddesamino-phenylalanine;

X₁₀ is aspartate-lysine dipeptide, a lysine-proline dipeptide, or aproline-lysine dipeptide;

X₁₁ is threonine, alanine, or a threonine-arginine-arginine tripeptide;

-   -   wherein R₁ is selected from the group consisting of H and C₁-C₈        alkyl; and    -   R₂ and R₄ are independently selected from the group consisting        of H, C₁-C₈ alkyl, C₂-C₈ alkenyl, (C₁-C₄ alkyl)OH, (C₁-C₄        alkyl)SH, (C₂-C₃ alkyl)SCH₃, (C₁-C₄ alkyl)CONH₂, (C₁-C₄        alkyl)COOH, (C₁-C₄ alkyl)NH₂, (C₁-C₄ alkyl)NHC(NH₂ ⁺)NH₂, (C₀-C₄        alkyl)(C₃-C₆ cycloalkyl), (C₀-C₄ alkyl)(C₆-C₁₀ aryl)R₇, and        CH₂(C₅-C₉ heteroaryl);    -   R₃ is selected from the group consisting of C₁-C₈ alkyl, (C₁-C₄        alkyl)OH, (C₁-C₄ alkyl)SH, (C₁-C₄ alkyl)NH₂, (C₃-C₆)cycloalkyl        or R₄ and R₃ together with the atoms to which they are attached        form a 5 or 6 member heterocyclic ring;    -   R₅ is NHR₆ or OH;    -   R₆ is H, or R₆ and R₂ together with the atoms to which they are        attached form a 5 or 6 member heterocyclic ring;    -   R₇ is selected from the group consisting of H and OH; and    -   R₁₃ and R₁₄ are independently COOH or CONH₂, with the proviso        that one and only one of X₁₂, X₁₃, or J is a dipeptide        comprising the general structure:

(i.e., only one dipeptide prodrug element is attached to the insulinpeptide). In one embodiment J is a dipeptide comprising the generalstructure:

and X and X₁₂ are each OH and X₁₃ is H, with the further proviso thatwhen R₄ and R₃ together with the atoms to which they are attached form a5 member heterocyclic ring, both R₁ and R₂ are other than H. In analternative embodiment X₁₂ is NHR₁₁, J and X₁₃ are each H and X is OH.In another alternative embodiment X₁₃ is NHR₁₂, X and X₁₂ are each OHand J is H. In one embodiment the B chain comprises the sequenceJ-X₉VNQX₄LCGX₅X₆LVEALX₇LVCGERGFX₈YTPKT (SEQ ID NO: 15) orJ-X₉VNQX₄LCGX₅X₆LVEALX₇LVCGERGFX₈YTKPT (SEQ ID NO: 16), wherein J, X₄,X₅, X₆, X₇, X₈ and X₉ are defined as immediately above. In a furtherembodiment R₃ is C₁-C₆ alkyl and R₄ is selected from the groupconsisting of H, C₁-C₄ alkyl, or R₃ and R₄ together with the atoms towhich they are attached form a 5 member heterocyclic ring. In anotherfurther embodiment X₄ is histidine, X₅ is serine and X₆ is histidine.

In another embodiment an insulin prodrug analog is provided comprisingan A chain sequence of Z-GIVEQCCX₁SICSLYQLENX₂CX₃ (SEQ ID NO: 3) and a Bchain sequence comprising a sequence of X₄LCGX₅X₆LVEALYLVCGERGFF (SEQ IDNO: 4) wherein

Z is H or a dipeptide comprising the general structure:

X₁ is selected from the group consisting of threonine and histidine;

X₂ is an amino acid of the general structure

-   -   wherein X is selected from the group consisting of OH, NH₂,        NHR₁₀ and OCH₃, wherein R₁₀ is a dipeptide comprising the        general structure:

X₃ is selected from the group consisting of asparagine, glycine,alanine, threonine, or serine;

X₄ is selected from the group consisting of histidine and threonine;

X₅ is selected from the group consisting of alanine, glycine and serine;

X₆ is selected from the group consisting of histidine, aspartic acid,glutamic acid, homocysteic acid and cysteic acid;

wherein R₁ is selected from the group consisting of H and C₁-C₈ alkyl;

-   -   R₂ and R₄ are independently selected from the group consisting        of H, C₁-C₈ alkyl, C₂-C₈ alkenyl, (C₁-C₄ alkyl)OH, (C₁-C₄        alkyl)SH, (C₂-C₃ alkyl)SCH₃, (C₁-C₄ alkyl)CONH₂, (C₁-C₄        alkyl)COOH, (C₁-C₄ alkyl)NH₂, (C₁-C₄ alkyl)NHC(NH₂ ⁺) NH₂,        (C₀-C₄ alkyl)(C₃-C₆ cycloalkyl), (C₀-C₄ alkyl)(C₆-C₁₀ aryl)R₇,        CH₂(C₅-C₉ heteroaryl), or R₁ and R₂ together with the atoms to        which they are attached form a C₃-C₆ cycloalkyl;    -   R₃ is selected from the group consisting of C₁-C₈ alkyl, (C₁-C₄        alkyl)OH, (C₁-C₄ alkyl)SH, (C₃-C₆)cycloalkyl or R₄ and R₃        together with the atoms to which they are attached form a 5 or 6        member heterocyclic ring;    -   R₅ is NHR₆ or OH;    -   R₆ is H, or R₆ and R₂ together with the atoms to which they are        attached form a 5 or 6 member heterocyclic ring; and    -   R₇ is selected from the group consisting of H and OH, with the        proviso that X and Z are not both dipeptides and Z is not H when        X is OH. In one embodiment when Z is a dipeptide comprising the        general structure:

and R₄ and R₃ together with the atoms to which they are attached form a5 member heterocyclic ring, then at least one of R₁ and R₂ are otherthan H. In one embodiment the A chain comprises a sequence ofZ-GIVEQCCX₁SICSLYQLENYCX₃ (SEQ ID NO: 17) and the B chain sequencecomprises the sequence X₉VNQX₄LCGX₅X₆LVEALYLVCGERGFFYTPKT (SEQ ID NO:12) or X₉VNQX₄LCGX₅X₆LVEALYLVCGERGFFYTKPT (SEQ ID NO: 13).

In an alternative embodiment an insulin prodrug analog is providedcomprising an A chain sequence of GIVEQCCX₁SICSLYQLENX₂CX₃ (SEQ ID NO:3) and a B chain sequence comprising the sequenceX₉VNQX₄LCGX₅X₆LVEALYLVCGERGFFYTPKT (SEQ ID NO: 12) orX₉VNQX₄LCGX₅X₆LVEALYLVCGERGFFYTKPT (SEQ ID NO: 13), wherein X₁ isselected from the group consisting of threonine and histidine;

X₂ is an amino acid of the general structure

-   -   wherein U is an amino acid or a hydroxyl acid and O is an        N-alkylated amino acid;

X₃ is asparagine, glycine, alanine, threonine, or serine;

X₄ is selected from the group consisting of histidine and threonine;

X₅ is selected from the group consisting of alanine, glycine and serine;

X₆ is selected from the group consisting of histidine, aspartic acid,glutamic acid, homocysteic acid and cysteic acid;

X₉ is selected from the group consisting of phenylalanine anddesamino-phenylalanine. In one embodiment U-O represent a dipeptide ofthe general structure;

wherein R₁ is selected from the group consisting of H and C₁-C₈ alkyl;

R₂ and R₄ are independently selected from the group consisting of H,C₁-C₈ alkyl, C₂-C₈ alkenyl, (C₁-C₄ alkyl)OH, (C₁-C₄ alkyl)SH, (C₂-C₃alkyl)SCH₃, (C₁-C₄ alkyl)CONH₂, (C₁-C₄ alkyl)COOH, (C₁-C₄ alkyl)NH₂,(C₁-C₄ alkyl)NHC(NH₂ ⁺) NH₂, (C₀-C₄ alkyl)(C₃-C₆ cycloalkyl), (C₀-C₄alkyl)(C₆-C₁₀ aryl)R₇, CH₂(C₅-C₉ heteroaryl), or R₁ and R₂ together withthe atoms to which they are attached form a C₃-C₆ cycloalkyl;

R₃ is selected from the group consisting of C₁-C₈ alkyl, (C₁-C₄alkyl)OH, (C₁-C₄ alkyl)SH, (C₁-C₄ alkyl)NH₂, (C₃-C₆)cycloalkyl or R₄ andR₃ together with the atoms to which they are attached form a 5 or 6member heterocyclic ring;

R₅ is NHR₆ or OH;

R₆ is H, or R₆ and R₂ together with the atoms to which they are attachedform a 5 or 6 member heterocyclic ring; and

R₇ is selected from the group consisting of H and OH. In a furtherembodiment X₇ is tyrosine, X₈ is phenylalanine and X₉ is phenylalanine,and in an additional further embodiment X₄ is histidine, X₅ is serineand X₆ is histidine. In a further embodiment U-O represent a dipeptideof the general structure;

wherein

R₁, R₂, R₄ and R₈ are independently selected from the group consistingof H, C₁-C₁₈ alkyl, C₂-C₁₈ alkenyl, (C₁-C₁₈ alkyl)OH, (C₁-C₁₈ alkyl)SH,(C₂-C₃ alkyl)SCH₃, (C₁-C₄ alkyl)CONH₂, (C₁-C₄ alkyl)COOH, (C₁-C₄alkyl)NH₂, (C₁-C₄ alkyl)NHC(NH₂ ⁺)NH₂, (C₀-C₄ alkyl)(C₃-C₆ cycloalkyl),(C₀-C₄ alkyl)(C₂-C₅ heterocyclic), (C₀-C₄ alkyl)(C₆-C₁₀ aryl)R₇, (C₁-C₄alkyl)(C₃-C₉ heteroaryl), and C₁-C₁₂ alkyl(W₁)C₁-C₁₂ alkyl, wherein W₁is a heteroatom selected from the group consisting of N, S and O, or R₁and R₂ together with the atoms to which they are attached form a C₃-C₁₂cycloalkyl; or R₄ and R₈ together with the atoms to which they areattached form a C₃-C₆ cycloalkyl;

R₃ is selected from the group consisting of C₁-C₁₈ alkyl, (C₁-C₁₈alkyl)OH, (C₁-C₁₈ alkyl)NH₂, (C₁-C₁₈ alkyl)SH, (C₀-C₄alkyl)(C₃-C₆)cycloalkyl, (C₀-C₄ alkyl)(C₂-C₅ heterocyclic), (C₀-C₄alkyl)(C₆-C₁₀ aryl)R₇, and (C₁-C₄ alkyl)(C₃-C₉ heteroaryl) or R₄ and R₃together with the atoms to which they are attached form a 4, 5 or 6member heterocyclic ring;

R₅ is NHR₆ or OH;

R₆ is H, C₁-C₈ alkyl or R₆ and R₁ together with the atoms to which theyare attached form a 4, 5 or 6 member heterocyclic ring; and

R₇ is selected from the group consisting of hydrogen, C₁-C₁₈ alkyl,C₂-C₁₈ alkenyl, (C₀-C₄ alkyl)CONH₂, (C₀-C₄ alkyl)COOH, (C₀-C₄ alkyl)NH₂,(C₀-C₄ alkyl)OH, and halo.

In another embodiment an insulin prodrug analog is provided comprisingan A chain sequence of Z-GIVEQCCTSICSLYQLENX₂CX₃-R₁₃ (SEQ ID NO: 18) anda B chain sequence comprising a sequence of X₄LCGSHLVEALYLVCGERGFF-R₁₄(SEQ ID NO: 19) wherein

Z is H or an amide linked dipeptide comprising the general structure:

X₂ is an amino acid of the general structure

-   -   wherein X is selected from the group consisting of OH and NHR₁₀;        -   wherein R₁₀ is a dipeptide comprising the general structure:

X₃ is serine, asparagine or glycine;

X₄ is selected from the group consisting of histidine and threonine;

R₁ is selected from the group consisting of H and C₁-C₈ alkyl;

R₂ and R₄ are independently selected from the group consisting of H,C₁-C₈ alkyl, C₂-C₈ alkenyl, (C₁-C₄ alkyl)OH, (C₁-C₄ alkyl)SH, (C₂-C₃alkyl)SCH₃, (C₁-C₄ alkyl)CONH₂, (C₁-C₄ alkyl)COOH, (C₁-C₄ alkyl)NH₂,(C₁-C₄ alkyl)NHC(NH₂ ⁺) NH₂, (C₀-C₄ alkyl)(C₃-C₆ cycloalkyl), (C₀-C₄alkyl)(C₆-C₁₀ aryl)R₇, CH₂(C₅-C₉ heteroaryl), or R₁ and R₂ together withthe atoms to which they are attached form a C₃-C₆ cycloalkyl;

R₃ is selected from the group consisting of C₁-C₈ alkyl, (C₁-C₄alkyl)OH, (C₁-C₄ alkyl)SH, (C₃-C₆)cycloalkyl or R₄ and R₃ together withthe atoms to which they are attached form a 5 or 6 member heterocyclicring;

R₅ is NHR₆ or OH;

R₆ is H, or R₆ and R₂ together with the atoms to which they are attachedform a 5 or 6 member heterocyclic ring;

R₇ is selected from the group consisting of H and OH; and

R₁₃ and R₁₄ are independently COOH or CONH₂; with the proviso that whenZ is H, X is not OH and when X is OH, Z is not H. In one embodiment R₁₃is COOH and R₁₄ is CONH₂. In a further embodiment X₂ is an amino acid ofthe general structure of Formula III:

Z is H, X₃ is serine, X₄ is histidine, R₁₃ is COOH and R₁₄ is CONH₂. Inan additional further embodiment

R₁ is selected from the group consisting of H and C₁-C₄ alkyl;

R₂ is selected from the group consisting of H, C₁-C₆ alkyl, C₂-C₈alkenyl, (C₁-C₄ alkyl)OH, (C₁-C₄ alkyl)NH₂, (C₀-C₄ alkyl)(C₃-C₆cycloalkyl), (C₀-C₄ alkyl)(C₆-C₁₀ aryl)R₇, and CH₂(C₅-C₉ heteroaryl) orR₂ and R₆ together with the atoms to which they are attached form a 5member heterocyclic ring;

R₃ is C₁-C₆ alkyl;

R₄ is selected from the group consisting of H and C₁-C₄ alkyl or R₃ andR₄ together with the atoms to which they are attached form a 5 memberheterocyclic ring. In a further embodiment R₃ is CH₃, R₄ is H and R₅ isNH₂, or alternatively, R₅ is NH₂ and R₃ and R₄ together with the atomsto which they are attached form a 5 member heterocyclic ring. Inaccordance with one embodiment the B chain of the insulin prodrug analogcomprises the sequence FVNQHLCGSHLVEALYLVCGERGFFYTPKT-R₁₄ (SEQ ID NO:8), FVNQHLCGSHLVEALYLVCGERGFFYTKPT-R₁₄ (SEQ ID NO: 9) orFVNQHLCGSHLVEALYLVCGERGFFYTPKTRR-R₁₄ (SEQ ID NO: 10), wherein R₁₄ isCOOH or CONH₂, and in one embodiment R₁₄ is CONH₂.

In one embodiment an insulin prodrug analog is provided comprising apolypeptide of the sequence Z-GIVEQCCX₁SICSLYQLENX₂CX₃ (SEQ ID NO: 3),wherein

Z is a dipeptide comprising the general structure:

X₁ is selected from the group consisting of threonine and histidine;

X₂ is an amino acid of the general structure

-   -   wherein X is selected from the group consisting of OH, NH₂, and        OCH₃;

X₃ is asparagine, glycine, alanine, threonine, or serine;

R₁ is selected from the group consisting of H and C₁-C₈ alkyl; and

R₂ and R₄ are independently selected from the group consisting of H,C₁-C₈ alkyl, C₂-C₈ alkenyl, (C₁-C₄ alkyl)OH, (C₁-C₄ alkyl)SH, (C₂-C₃alkyl)SCH₃, (C₁-C₄ alkyl)CONH₂, (C₁-C₄ alkyl)COOH, (C₁-C₄ alkyl)NH₂,(C₁-C₄ alkyl)NHC(NH₂ ⁺) NH₂, (C₀-C₄ alkyl)(C₃-C₆ cycloalkyl), (C₀-C₄alkyl)(C₆-C₁₀ aryl)R₇, CH₂(C₅-C₉ heteroaryl), or R₁ and R₂ together withthe atoms to which they are attached form a C₃-C₆ cycloalkyl;

R₃ is selected from the group consisting of C₁-C₈ alkyl, (C₁-C₄alkyl)OH, (C₁-C₄ alkyl)SH, (C₁-C₄ alkyl)NH₂, (C₃-C₆)cycloalkyl or R₄ andR₃ together with the atoms to which they are attached form a 5 or 6member heterocyclic ring;

R₅ is NHR₆ or OH;

R₆ is H, or R₆ and R₂ together with the atoms to which they are attachedform a 5 or 6 member heterocyclic ring; and

R₇ is selected from the group consisting of H and OH. In one embodimentX₁ is threonine and X₃ is asparagine or glycine and in a furtherembodiment R₃ is C₁-C₆ alkyl and R₄ is selected from the groupconsisting of H, C₁-C₄ alkyl, (C₃-C₆)cycloalkyl, (C₁-C₄ alkyl)OH, (C₁-C₄alkyl)SH and (C₀-C₄ alkyl)(C₆ aryl)R₇, or R₃ and R₄ together with theatoms to which they are attached form a 5 member heterocyclic ring, withthe proviso that when R₄ and R₃ together with the atoms to which theyare attached form a 5 or 6 member heterocyclic ring, both R₁ and R₂ areboth other than H.

In one embodiment an insulin prodrug analog is provided comprising apolypeptide of the sequence GIVEQCCX₁SICSLYQLENX₂CX₃ (SEQ ID NO: 3),wherein

X₁ is selected from the group consisting of threonine and histidine;

X₂ is an amino acid of the general structure

X₃ is asparagine, glycine, alanine, threonine, or serine;

R₁ is selected from the group consisting of H and C₁-C₈ alkyl; and

R₂ and R₄ are independently selected from the group consisting of H,C₁-C₈ alkyl, C₂-C₈ alkenyl, (C₁-C₄ alkyl)OH, (C₁-C₄ alkyl)SH, (C₂-C₃alkyl)SCH₃, (C₁-C₄ alkyl)CONH₂, (C₁-C₄ alkyl)COOH, (C₁-C₄ alkyl)NH₂,(C₁-C₄ alkyl)NHC(NH₂ ⁺) NH₂, (C₀-C₄ alkyl)(C₃-C₆ cycloalkyl), (C₀-C₄alkyl)(C₆-C₁₀ aryl)R₇, CH₂(C₅-C₉ heteroaryl), or R₁ and R₂ together withthe atoms to which they are attached form a C₃-C₆ cycloalkyl;

R₃ is selected from the group consisting of C₁-C₈ alkyl, (C₁-C₄alkyl)OH, (C₁-C₄ alkyl)SH, (C₃-C₆)cycloalkyl or R₄ and R₃ together withthe atoms to which they are attached form a 5 or 6 member heterocyclicring;

R₅ is NHR₆ or OH;

R₆ is H, or R₆ and R₂ together with the atoms to which they are attachedform a 5 or 6 member heterocyclic ring; and

R₇ is selected from the group consisting of H and OH. In one embodimentthe A chain of GIVEQCCX₁SICSLYQLENX₂CX₃ (SEQ ID NO: 3) as definedimmediately above is linked, either by disulfide bonds or as a singlechain polypeptide, to a B chain comprising the sequenceX₁₄-X₄LCGX₅X₆LVEALX₇LVCGERGFX₈ (SEQ ID NO: 14) wherein

X₄ is selected from the group consisting of histidine and threonine;

X₅ is selected from the group consisting of alanine, glycine and serine;

X₆ is selected from the group consisting of histidine, aspartic acid,glutamic acid, homocysteic acid and cysteic acid;

X₇ is tyrosine; and

X₈ is phenylalanine.

In a further embodiment the dipeptide prodrug element has the structureof Formula I wherein

R₁, R₂, and R₄ are independently selected from the group consisting ofH, C₁-C₁₈ alkyl, C₂-C₁₈ alkenyl, (C₁-C₁₈ alkyl)OH, (C₁-C₁₈ alkyl)SH,(C₂-C₃ alkyl)SCH₃, (C₁-C₄ alkyl)CONH₂, (C₁-C₄ alkyl)COOH, (C₁-C₄alkyl)NH₂, (C₁-C₄ alkyl)NHC(NH₂ ⁺)NH₂, (C₀-C₄ alkyl)(C₃-C₆ cycloalkyl),(C₀-C₄ alkyl)(C₂-C₅ heterocyclic), (C₀-C₄ alkyl)(C₆-C₁₀ aryl)R₇, (C₁-C₄alkyl)(C₃-C₉ heteroaryl), and C₁-C₁₂ alkyl(W₁)C₁-C₁₂ alkyl, wherein W₁is a heteroatom selected from the group consisting of N, S and O, or R₁and R₂ together with the atoms to which they are attached form a C₃-C₁₂cycloalkyl;

R₃ is selected from the group consisting of C₁-C₁₈ alkyl, (C₁-C₁₈alkyl)OH, (C₁-C₁₈ alkyl)NH₂, (C₁-C₁₈ alkyl)SH, (C₀-C₄alkyl)(C₃-C₆)cycloalkyl, (C₀-C₄ alkyl)(C₂-C₅ heterocyclic), (C₀-C₄alkyl)(C₆-C₁₀ aryl)R₇, and (C₁-C₄ alkyl)(C₃-C₉ heteroaryl) or R₄ and R₃together with the atoms to which they are attached form a 4, 5 or 6member heterocyclic ring;

R₅ is NHR₆ or OH;

R₆ is H, C₁-C₈ alkyl or R₆ and R₁ together with the atoms to which theyare attached form a 4, 5 or 6 member heterocyclic ring; and

R₇ is selected from the group consisting of hydrogen, C₁-C₁₈ alkyl,C₂-C₁₈ alkenyl, (C₀-C₄ alkyl)CONH₂, (C₀-C₄ alkyl)COOH, (C₀-C₄ alkyl)NH₂,(C₀-C₄ alkyl)OH, and halo. In a further embodiment R₃ is C₁-C₆ alkyl andR₄ is selected from the group consisting of H and C₁-C₄ alkyl, oralternatively, R₃ and R₄ together with the atoms to which they areattached form a 5 member heterocyclic ring. In one embodiment X₁ isthreonine and X₃ is asparagine or glycine.

In one embodiment R₁ is selected from the group consisting of H andC₁-C₆ alkyl, R₂ is selected from the group consisting of H, C₁-C₆ alkyl,(C₁-C₄ alkyl)C(O)NH₂, CH₂OH, (C₁-C₄ alkyl)NH₂, (C₃-C₆ cycloalkyl) andCH₂(C₆ aryl)R₇ or R₂ and R₆ together with the atoms to which they areattached form a 5 member heterocyclic ring;

-   -   R₃ is C₁-C₆ alkyl;    -   R₄ is selected from the group consisting of H, C₁-C₄ alkyl,        (C₃-C₆)cycloalkyl, and (C₀-C₄ alkyl)(C₆-C₁₀ aryl)R₇, or R₃ and        R₄ together with the atoms to which they are attached form a 5        member heterocyclic ring    -   R₅ is NHR₆ or OH;    -   R₆ is H, or R₆ and R₂ together with the atoms to which they are        attached form a 5 or 6 member heterocyclic ring; and    -   R₇ is selected from the group consisting of H and OH.

In accordance with one embodiment a single-chain insulin prodrug analogis provided wherein the carboxy terminus of the human insulin B chain,or a functional analog thereof, is covalently linked to the N-terminusof the human insulin A chain, or functional analog thereof, and furtherwherein a dipeptide prodrug moiety having the general structure:

is covalently bound at the N-terminus of the peptide, or at the sidechain of an amino acid via an amide bond, including for example atpositions corresponding to A19, B16 or B25 of the respective nativeinsulin A chain or B chain. In accordance with one embodiment thesingle-chain insulin analog comprises a compound of the formula: B-P-A,wherein: B represents the B-chain of human insulin or one of thefunctional or prodrug analogs of a B chain as disclosed herein, Arepresents the A chain of human insulin or one of the functional orprodrug analogs of an A chain as disclosed herein, and P represents alinker, including a peptide linker, that covalently joins the A chain tothe B chain. In one embodiment the linker is a peptide linker of about 5to about 18, or about 10 to about 14, or about 4 to about 8, or about 6amino acids. In one embodiment the B chain is linked to the A chain viapeptide linker of 4-12 or 4-8 amino acids.

In one embodiment the single-chain insulin prodrug analog of the formulaB-P-A comprises an A chain having the sequence GIVEQCCX₁SICSLYQLENX₂CX₃(SEQ ID NO: 3) and a B chain sequence that includes the sequence ofJ-X₁₄-X₄LCGX₅X₆LVEALX₇LVCGERGFX₈ (SEQ ID NO: 14) wherein J is H or adipeptide comprising the general structure:

wherein X₁₄ is either a bond or a 1 to 4 amino acid sequence selectedfrom the group consisting of a FVNQ (SEQ ID NO: 11), VNQ, NQ and Q.

X₁ is selected from the group consisting of threonine and histidine;

X₂ is an amino acid of the general structure

-   -   wherein X is selected from the group consisting of OH, NHR₁₀ and        OCH₃; wherein R₁₀ is H or a dipeptide comprising the general        structure:

X₃ is asparagine, glycine, alanine, threonine, or serine.

X₄ is selected from the group consisting of histidine and threonine;

X₅ is selected from the group consisting of alanine, glycine and serine;

X₆ is selected from the group consisting of histidine, aspartic acid,glutamic acid, homocysteic acid and cysteic acid;

X₇ is an amino acid of the general structure

-   -   wherein X₁₂ is selected from the group consisting of OH, OCH₃        and NHR₁₁, wherein R₁₁ is H or a dipeptide comprising the        general structure:

X₈ is an amino acid of the general structure

-   -   wherein X₁₃ is selected from the group consisting of H, OH, OCH₃        and NHR₁₂, wherein R₁₂ is H or a dipeptide comprising the        general structure:

-   -   wherein R₁ is selected from the group consisting of H and C₁-C₈        alkyl; and    -   R₂ and R₄ are independently selected from the group consisting        of H, C₁-C₈ alkyl, C₂-C₈ alkenyl, (C₁-C₄ alkyl)OH, (C₁-C₄        alkyl)SH, (C₂-C₃ alkyl)SCH₃, (C₁-C₄ alkyl)CONH₂, (C₁-C₄        alkyl)COOH, (C₁-C₄ alkyl)NH₂, (C₁-C₄ alkyl)NHC(NH₂ ⁺) NH₂,        (C₀-C₄ alkyl)(C₃-C₆ cycloalkyl), (C₀-C₄ alkyl)(C₆-C₁₀ aryl)R₇,        and CH₂(C₅-C₉ heteroaryl);    -   R₃ is selected from the group consisting of C₁-C₈ alkyl, (C₁-C₄        alkyl)OH, (C₁-C₄ alkyl)SH, (C₁-C₄ alkyl)NH₂, (C₃-C₆)cycloalkyl        or R₄ and R₃ together with the atoms to which they are attached        form a 5 or 6 member heterocyclic ring;    -   R₅ is NHR₆ or OH;    -   R₆ is H, or R₆ and R₂ together with the atoms to which they are        attached form a 5 or 6 member heterocyclic ring; and    -   R₇ is selected from the group consisting of H and OH, with the        proviso that one and only one of Z, J, R₁₀, R₁₁ or R₁₂ is a        dipeptide comprising the general structure:

(i.e., only one dipeptide prodrug element is attached to the insulinpeptide).

In one embodiment J is a dipeptide comprising the general structure:

and X and X₁₂ are each OH and X₁₃ is H, with the proviso that when R₄and R₃ together with the atoms to which they are attached form a 5member heterocyclic ring, both R₁ and R₂ are other than H. In analternative embodiment X₁₂ comprises the dipeptide of Formula I, J andX₁₃ are each H and X is OH. In another alternative embodiment X₁₃ iscomprises the dipeptide of Formula I, X and X₁₂ are each OH and J is H.In another embodiment X comprises the dipeptide of Formula I, J and X₁₃are each H and X₁₂ is OH. In one embodiment the B chain comprises thesequence J-X₉VNQX₄LCGX₅X₆LVEALX₇LVCGERGFX₈YTPKT (SEQ ID NO: 15) orJ-X₉VNQX₄LCGX₅X₆LVEALX₇LVCGERGFX₈YTKPT (SEQ ID NO: 16), wherein J, X₄,X₅, X₆, X₇, X₈ and X₉ are defined as immediately above. In a furtherembodiment R₃ is C₁-C₆ alkyl and R₄ is selected from the groupconsisting of H, C₁-C₄ alkyl, or R₃ and R₄ together with the atoms towhich they are attached form a 5 member heterocyclic ring. In anotherfurther embodiment X₄ is histidine, X₅ is serine and X₆ is histidine.

In one embodiment the single chain insulin analog comprises a compoundof the formula: B-P-A, wherein:

B represents a B chain sequence comprising a sequence ofX₄LCGX₅X₆LVEALYLVCG ERGFF (SEQ ID NO: 4) or a functional analog thereof,

A represents an A chain sequence comprising a sequence ofGIVEQCCX₁SICSLYQLENX₂CX₃ (SEQ ID NO: 3) or a functional analog thereof,wherein

X₁ is selected from the group consisting of threonine and histidine;

X₂ is an amino acid of the general structure

X₃ is asparagine or glycine;

X₄ is selected from the group consisting of histidine and threonine;

X₅ is selected from the group consisting of alanine, glycine and serine;

X₆ is selected from the group consisting of histidine, aspartic acid,glutamic acid, homocysteic acid and cysteic acid.

P represents a linker, including a peptide linker, that covalently joinsthe amino-terminus of the A chain to the carboxy-terminus of the Bchain. In an alternative embodiment the single-chain insulin analogcomprises a compound of the formula: A-P-B, wherein: A represents ahuman insulin A chain, or a functional analog thereof, B represents ahuman insulin B chain, or a functional analog thereof, and P representsa linker, including a peptide linker, that covalently joins theamino-terminus of the B chain to the carboxy-terminus of the A chain. Inone embodiment the peptide linker comprises 4 to 8 amino acids.

In accordance with one embodiment the peptide linker is 5 to 18 aminoacids in length and comprises a sequence selected from the groupconsisting of: Gly-Gly-Gly-Pro-Gly-Lys-Arg (SEQ ID NO: 22),Gly-Tyr-Gly-Ser-Ser-Ser-Arg-Arg-Ala-Pro-Gln-Thr (SEQ ID NO: 23),Arg-Arg-Gly-Pro-Gly-Gly-Gly (SEQ ID NO: 32), Gly-Gly-Gly-Gly-Gly-Lys-Arg(SEQ ID NO: 24), Arg-Arg-Gly-Gly-Gly-Gly-Gly (SEQ ID NO: 25),Gly-Gly-Ala-Pro-Gly-Asp-Val-Lys-Arg (SEQ ID NO: 26),Arg-Arg-Ala-Pro-Gly-Asp-Val-Gly-Gly (SEQ ID NO: 27),Gly-Gly-Tyr-Pro-Gly-Asp-Val-Lys-Arg (SEQ ID NO: 28),Arg-Arg-Tyr-Pro-Gly-Asp-Val-Gly-Gly (SEQ ID NO: 29),Gly-Gly-His-Pro-Gly-Asp-Val-Lys-Arg (SEQ ID NO: 30) andArg-Arg-His-Pro-Gly-Asp-Val-Gly-Gly (SEQ ID NO: 31). In one embodimentthe peptide linker is 7 to 12 amino acids in length and comprises thesequence Gly-Gly-Gly-Pro-Gly-Lys-Arg (SEQ ID NO: 22) orGly-Tyr-Gly-Ser-Ser-Ser-Arg-Arg-Ala-Pro-Gln-Thr (SEQ ID NO: 23).

In a further embodiment the peptide linker comprises a sequence selectedfrom the group consisting of AGRGSGK (SEQ ID NO: 35), AGLGSGK (SEQ IDNO: 36), AGMGSGK (SEQ ID NO: 37), ASWGSGK (SEQ ID NO: 38), TGLGSGQ (SEQID NO: 39), TGLGRGK (SEQ ID NO: 40), TGLGSGK (SEQ ID NO: 41), HGLYSGK(SEQ ID NO: 42), KGLGSGQ (SEQ ID NO: 43), VGLMSGK (SEQ ID NO: 44),VGLSSGQ (SEQ ID NO: 45), VGLYSGK (SEQ ID NO: 46), VGLSSGK (SEQ ID NO:47), VGMSSGK (SEQ ID NO: 483, VWSSSGK (SEQ ID NO: 49), VGSSSGK (SEQ IDNO: 50). VGMSSGK (SEQ ID NO: 51), TGLGSGR (SEQ ID NO: 52), TGLGKGQ (SEQID NO: 53), KGLSSGQ (SEQ ID NO: 54), VKLSSGQ (SEQ ID NO: 55), VGLKSGQ(SEQ ID NO: 56), TGLGKGQ (SEQ ID NO: 57) SRVSRRSR (SEQ ID NO: 65),GYGSSSRRAPQT (SEQ ID NO: 66) and VGLSKGQ (SEQ ID NO: 58). In oneembodiment the linker comprises GSSSRRAP (SEQ ID NO: 67) or SRVSRRSR(SEQ ID NO: 65)

In one embodiment the single-chain insulin analog has the amino acidsequence:Phe-Val-Asn-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-Tyr-Leu-Val-Cys-Gly-Glu-Arg-Gly-Phe-Phe-Tyr-Thr-Pro-Lys-Thr-Gly-Ile-Val-Glu-Gln-Cys-Cys-Thr-Ser-Ile-Cys-Ser-Leu-Tyr-Gln-Leu-Glu-Asn-Xaa-Cys-Asn(SEQ ID NO: 33) orPhe-Val-Asn-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-Tyr-Leu-Val-Cys-Gly-Glu-Arg-Gly-Phe-Phe-Tyr-Thr-Pro-Lys-Thr-Gln-Pro-Leu-Ala-Leu-Glu-Gly-Ser-Leu-Gln-Lys-Arg-Gly-Ile-Val-Glu-Gln-Cys-Cys-Thr-Ser-Ile-Cys-Ser-Leu-Tyr-Gln-Leu-Glu-Asn-Xaa-Cys-Asn(SEQ ID NO: 34) wherein Xaa is an amino acid of the general structure

The insulin peptides disclosed herein may be part of a dimer, trimer orhigher order multimer comprising at least two, three, or more peptidesbound via a linker, wherein at least one or both peptides comprises adipeptide prodrug element linked to the insulin peptide. The dimer maybe a homodimer or heterodimer, comprising peptides selected from thegroup consisting of native insulin, native IGF-1, native IGF-II andinsulin analog peptides as disclosed herein. In one embodiment, thelinker is selected from the group consisting of a bifunctional thiolcrosslinker and a bifunctional amine crosslinker. In certainembodiments, the linker is PEG, e.g., a 5 kDa PEG, 20 kDa PEG. In someembodiments, the linker is a disulfide bond.

For example, each monomer of the dimer may comprise a Cys residue (e.g.,a terminal or internally positioned Cys) and the sulfur atom of each Cysresidue participates in the formation of the disulfide bond. Eachmonomer of the dimer represents a heterodimer of an A and B chain. The Aand B chain are either linked via disulfide bonds or are prepared assingle chain peptides. In some aspects of the invention, the monomersare connected via terminal amino acids (e.g., N-terminal or C-terminal),via internal amino acids, or via a terminal amino acid of at least onemonomer and an internal amino acid of at least one other monomer. Inspecific aspects, the monomers are not connected via an N-terminal aminoacid. In some aspects, the monomers of the multimer are attachedtogether in a “tail-to-tail” orientation in which the C-terminal aminoacids of each monomer are attached together. A conjugate moiety may becovalently linked to any of the insulin peptides described herein,including a dimer, trimer or higher order multimer.

The prodrugs disclosed herein can be further modified to improve thepeptide's solubility in aqueous solutions at physiological pH, whileenhancing the effective duration of the peptide by preventing renalclearance of the peptide. Peptides are easily cleared because of theirrelatively small molecular size when compared to plasma proteins.Increasing the molecular weight of a peptide above 40 kDa exceeds therenal threshold and significantly extends duration in the plasma.Accordingly, in one embodiment the peptide prodrugs are further modifiedto comprise a covalently linked hydrophilic moiety. In one embodimentthe hydrophilic moiety is a plasma protein polyethylene oxide chain orthe Fc portion of an immunoglobin. Therefore, in one embodiment thepresently disclosed prodrugs are further modified to comprise one ormore hydrophilic groups covalently linked to the side chains of aminoacids.

In accordance with one embodiment the insulin prodrugs disclosed hereinare further modified by linking a hydrophilic moiety to either theN-terminal amino acid of the B chain or to the side chain of a lysineamino acid located at the carboxy terminus of the B chain, including forexample, at position 28 of SEQ ID NO: 9/SEQ ID NO: 13 or at position 29of SEQ ID NO: 8/SEQ ID NO: 12. In one embodiment a single-chain insulinprodrug analog is provided wherein one of the amino acids of the peptidelinker is modified by linking a hydrophilic moiety to the side chain ofthe peptide linker. In one embodiment the modified amino acid iscysteine, lysine or acetyl phenylalanine. In one embodiment the peptidelinker is selected from the group consisting of TGLGSGQ (SEQ ID NO: 39),VGLSSGQ (SEQ ID NO: 45), VGLSSGK (SEQ ID NO: 47), TGLGSGR (SEQ ID NO:52), TGLGKGQ (SEQ ID NO: 53), KGLSSGQ (SEQ ID NO: 54), VKLSSGQ (SEQ IDNO: 55), VGLKSGQ (SEQ ID NO: 56), TGLGKGQ (SEQ ID NO: 57) and VGLSKGQ(SEQ ID NO: 58) and the hydrophilic moiety (e.g., polyethylene glycol)is linked to the lysine side chain of the peptide linker.

In another embodiment the insulin prodrug analogs disclosed herein arefurther modified by the addition of a modified amino acid to the carboxyterminus of the B chain of the insulin prodrug, wherein the C-terminallyadded amino acid is modified to comprise a hydrophilic moiety linked tothe amino acid. In one embodiment the amino acid added to the C-terminusis a modified cysteine, lysine or acetyl phenylalanine. In oneembodiment the hydrophilic moiety is selected from the group consistingof a plasma protein, polyethylene oxide chain and an Fc portion of animmunoglobin.

In one embodiment the hydrophilic group is a polyethylene oxide chain,and in one embodiment two or more polyethylene oxide chains arecovalently attached to two or more amino acid side chains of the insulinprodrug analog. In accordance with one embodiment the hydrophilic moietyis covalently attached to an amino acid side chain of an insulin prodruganalog disclosed herein at a position selected from the group consistingof A9, A14, A15, B22, B28, B29 and the C-terminus or N-terminus of the Bchain. For insulin prodrug analogs having multiple polyethylene oxidechains, the polyethylene oxide chains can be attached at the N-terminalamino acid of the B chain or to the side chain of a lysine amino acidlocated at the carboxy terminus of the B chain, or by the addition of asingle amino acid at the C-terminus of the peptide wherein the addedamino acid has a polyethylene oxide chain linked to its side chain. Inaccordance with one embodiment the polyethylene oxide chain or otherhydrophilic moiety is linked to the side chain of one of the two aminoacids comprising the dipeptide prodrug element. In one embodiment thedipeptide prodrug element comprises a lysine (in the D or Lconfiguration) with a polyethylene oxide chain attached to the sidechain amine of the lysine.

Linkage of Hydrophilic Moieties

In another embodiment the solubility of the insulin analogs disclosedherein are enhanced by the covalent linkage of a hydrophilic moiety tothe peptide. Hydrophilic moieties can be attached to the insulin analogsunder any suitable conditions used to react a protein with an activatedpolymer molecule. Any means known in the art can be used, including viaacylation, reductive alkylation, Michael addition, thiol alkylation orother chemoselective conjugation/ligation methods through a reactivegroup on the PEG moiety (e.g., an aldehyde, amino, ester, thiol,α-haloacetyl, maleimido or hydrazino group) to a reactive group on thetarget compound (e.g., an aldehyde, amino, ester, thiol, α-haloacetyl,maleimido or hydrazino group). Activating groups which can be used tolink the water soluble polymer to one or more proteins include withoutlimitation sulfone, maleimide, sulfhydryl, thiol, triflate, tresylate,azidirine, oxirane and 5-pyridyl. If attached to the peptide byreductive alkylation, the polymer selected should have a single reactivealdehyde so that the degree of polymerization is controlled. See, forexample, Kinstler et al., Adv. Drug. Delivery Rev. 54: 477-485 (2002);Roberts et al., Adv. Drug Delivery Rev. 54: 459-476 (2002); and Zalipskyet al., Adv. Drug Delivery Rev. 16: 157-182 (1995).

Suitable hydrophilic moieties include polyethylene glycol (PEG),polypropylene glycol, polyoxyethylated polyols (e.g., POG),polyoxyethylated sorbitol, polyoxyethylated glucose, polyoxyethylatedglycerol (POG), polyoxyalkylenes, polyethylene glycol propionaldehyde,copolymers of ethylene glycol/propylene glycol, monomethoxy-polyethyleneglycol, mono-(C1-C10) alkoxy- or aryloxy-polyethylene glycol,carboxymethylcellulose, polyacetals, polyvinyl alcohol (PVA), polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene/maleicanhydride copolymer, poly(.beta.-amino acids) (either homopolymers orrandom copolymers), poly(n-vinyl pyrrolidone)polyethylene glycol,propropylene glycol homopolymers (PPG) and other polyakylene oxides,polypropylene oxide/ethylene oxide copolymers, colonic acids or otherpolysaccharide polymers, Ficoll or dextran and mixtures thereof.

The hydrophilic moiety, e.g., polyethylene glycol chain in accordancewith one embodiment has a molecular weight selected from the range ofabout 500 to about 40,000 Daltons. In one embodiment the hydrophilicmoiety, e.g. PEG, has a molecular weight selected from the range ofabout 500 to about 5,000 Daltons, or about 1,000 to about 5,000 Daltons.In another embodiment the hydrophilic moiety, e.g., PEG, has a molecularweight of about 10,000 to about 20,000 Daltons. In yet other exemplaryembodiments the hydrophilic moiety, e.g., PEG, has a molecular weight ofabout 20,000 to about 40,000 Daltons.

In one embodiment dextrans are used as the hydrophilic moiety. Dextransare polysaccharide polymers of glucose subunits, predominantly linked byα1-6 linkages. Dextran is available in many molecular weight ranges,e.g., about 1 kD to about 100 kD, or from about 5, 10, 15 or 20 kD toabout 20, 30, 40, 50, 60, 70, 80 or 90 kD.

Linear or branched polymers are contemplated. Resulting preparations ofconjugates may be essentially monodisperse or polydisperse, and may haveabout 0.5, 0.7, 1, 1.2, 1.5 or 2 polymer moieties per peptide.

In accordance with one embodiment, the insulin prodrug analogs disclosedherein are further modified by amino acid substitutions, wherein thesubstituting amino acid comprises a side chain suitable for crosslinkingwith hydrophilic moieties, including for example, polyethylene glycol.In one embodiment the amino acid at the position of the insulin prodruganalog where the hydrophilic moiety is to be linked is substituted (oradded at the C-terminus) with a natural or synthetic amino acid tointroduce, or allow for ease in attaching, the hydrophilic moiety. Forexample, in one embodiment a native amino acid at position selected fromA5, A8, A9, A10, A12, A14, A15, A17, A18, B1, B2, B3, B4, B5, B13, B14,B17, B21, B22, B26, B27, B28, B29 and B30 is substituted with a lysine,cysteine or acetyl phenylalanine residue (or a lysine, cysteine oracetyl phenylalanine residue is added to the C-terminus) to allow forthe covalent attachment of a polyethylene glycol chain.

In one embodiment the insulin prodrug analog has a single cysteineresidue added to the carboxy terminus of the B chain, or the insulinprodrug analog is substituted with at least one cysteine residue,wherein the side chain of the cysteine residue is further modified witha thiol reactive reagent, including for example, maleimido, vinylsulfone, 2-pyridylthio, haloalkyl, and haloacyl. These thiol reactivereagents may contain carboxy, keto, hydroxyl, and ether groups as wellas other hydrophilic moieties such as polyethylene glycol units. In analternative embodiment, the insulin prodrug analog has a single lysineresidue added to the carboxy terminus of the B chain, or the insulinprodrug analog is substituted with lysine, and the side chain of thesubstituting lysine residue is further modified using amine reactivereagents such as active esters (succinimido, anhydride, etc) ofcarboxylic acids or aldehydes of hydrophilic moieties such aspolyethylene glycol.

In those embodiments wherein the insulin prodrug analog comprises apolyethylene glycol chain, the polyethylene glycol chain may be in theform of a straight chain or it may be branched. In accordance with oneembodiment the polyethylene glycol chain has an average molecular weightselected from the range of about 20,000 to about 60,000 Daltons.Multiple polyethylene glycol chains can be linked to the insulin prodruganalog to provide an insulin prodrug analog with optimal solubility andblood clearance properties. In one embodiment the insulin prodrug analogis linked to a single polyethylene glycol chain that has an averagemolecular weight selected from the range of about 20,000 to about 60,000Daltons. In another embodiment the insulin prodrug analog is linked totwo polyethylene glycol chains wherein the combined average molecularweight of the two chains is selected from the range of about 40,000 toabout 80,000 Daltons. In one embodiment a single polyethylene glycolchain having an average molecular weight of 20,000 or 60,000 Daltons islinked to the insulin prodrug analog. In another embodiment a singlepolyethylene glycol chain is linked to the insulin prodrug analog andhas an average molecular weight selected from the range of about 40,000to about 50,000 Daltons. In one embodiment two polyethylene glycolchains are linked to the insulin prodrug analog wherein the first andsecond polyethylene glycol chains each have an average molecular weightof 20,000 Daltons. In another embodiment two polyethylene glycol chainsare linked to the insulin prodrug analog wherein the first and secondpolyethylene glycol chains each have an average molecular weight of40,000 Daltons.

In a further embodiment an insulin prodrug analog comprising two or morepolyethylene glycol chains covalently bound to the peptide is provided,wherein the total molecular weight of the polyethylene glycol chains isabout 40,000 to about 60,000 Daltons. In one embodiment the pegylatedinsulin prodrug analog comprises a polyethylene glycol chain linked toone or more amino acids selected from the N-terminus of the B chainand/or position 28 of SEQ ID NO: 9 or at position 29 of SEQ ID NO: 8,wherein the combined molecular weight of the PEG chain(s) is about40,000 to about 80,000 Daltons.

In accordance with one embodiment, an insulin peptide, or prodrug/depotderivative thereof, is fused to an accessory peptide which is capable offorming an extended conformation similar to chemical PEG (e.g., arecombinant PEG (rPEG) molecule), such as those described inInternational Patent Application Publication No. WO2009/023270 and U.S.Patent Application Publication No. US2008/0286808. The rPEG molecule isnot polyethylene glycol. The rPEG molecule in some aspects is apolypeptide comprising one or more of glycine, serine, glutamic acid,aspartic acid, alanine, or proline. In some aspects, the rPEG is ahomopolymer, e.g., poly-glycine, poly-serine, poly-glutamic acid,poly-aspartic acid, poly-alanine, or poly-proline. In other embodiments,the rPEG comprises two types of amino acids repeated, e.g.,poly(Gly-Ser), poly(Gly-Glu), poly(Gly-Ala), poly(Gly-Asp),poly(Gly-Pro), poly(Ser-Glu), etc. In some aspects, the rPEG comprisesthree different types of amino acids, e.g., poly(Gly-Ser-Glu). Inspecific aspects, the rPEG increases the half-life of the insulinpeptide. In some aspects, the rPEG comprises a net positive or netnegative charge. The rPEG in some aspects lacks secondary structure. Inone embodiment, the rPEG is greater than or equal to 10 amino acids inlength, and in one embodiment is about 40 to about 50 amino acids inlength. The accessory peptide in some aspects is fused to the N- orC-terminus of the peptide of the invention through a peptide bond or aproteinase cleavage site, or is inserted into the loops of the peptideof the invention. The rPEG in some aspects comprises an affinity tag oris linked to a PEG that is greater than 5 kDa. In one embodiment, therPEG confers the peptide of the invention with an increased hydrodynamicradius, serum half-life, protease resistance, or solubility and in someaspects confers the peptide with decreased immunogenicity.

In accordance with one embodiment, an insulin prodrug analog is providedwherein a plasma protein has been covalently linked to an amino acidside chain of the peptide to improve the solubility, stability and/orpharmacokinetics of the insulin prodrug analog. For example, serumalbumin can be covalently bound to the insulin prodrug analogs presentedherein. In one embodiment the plasma protein is covalently bound to theN-terminus of the B chain and/or to an amino acid corresponding toposition 28 of SEQ ID NO: 9 or at position 29 of SEQ ID NO: 8.

In accordance with one embodiment, an insulin prodrug analog is providedwherein a linear amino acid sequence representing the Fc portion of animmunoglobin molecule has been covalently linked to an amino acid sidechain of an insulin prodrug analog disclosed herein to improve thesolubility, stability and/or pharmacokinetics of the insulin prodruganalog. For example, the amino acid sequence representing the Fc portionof an immunoglobin molecule can be covalently bound to the N-terminus ofthe B chain or the C-terminus of the A or B chain, or the C-terminus ofan A or B chain that has been terminally extended. For example, theamino acid sequence representing the Fc portion of an immunoglobinmolecule can be covalently bound to the C-terminus of the B chain,including for example linkage to an amino acid corresponding to position28 of SEQ ID NO: 9 or at position 29 of SEQ ID NO: 8. The Fc portion istypically one isolated from IgG, but the Fc peptide fragment from anyimmunoglobin should function equivalently.

In a specific aspect of the invention, the insulin prodrug analog ismodified to comprise an alkyl or acyl group by direct alkylation oracylation of an amine, hydroxyl, or thiol of a side chain of an aminoacid of the insulin prodrug analog. In one embodiment, the insulinprodrug analog is directly acylated through the side chain amine,hydroxyl, or thiol of an amino acid. In one embodiment, acylation is atone or more positions selected from A9, A14, A15, B22, B28 or B29. Inthis regard, the acylated insulin prodrug analog can comprise an A chainamino acid sequence of SEQ ID NO: 3 and a B chain of SEQ ID NO: 5, or amodified amino acid sequence of SEQ ID NO: 3 and/or SEQ ID NO: 5 with atleast one of the amino acids at positions A9, A14, A15, B22, B28 or B29modified to any amino acid comprising a side chain amine, hydroxyl, orthiol. In some specific embodiments, the direct acylation of the insulinprodrug analog occurs through the side chain amine, hydroxyl, or thiolof the amino acid at position B28 or B29. In one further embodiment theinsulin prodrug analog comprises an acyl group of a carboxylic acid with1-24 carbon atoms bound to the epsilon-amino group of a Lys present atposition B28 or B29. In one embodiment a single-chain insulin prodruganalog is provided wherein one of the amino acids of the peptide linkeris modified to comprise an acyl group by direct acylation of an amine,hydroxyl, or thiol of a side chain of an amino acid of the peptidelinker. In accordance with one embodiment the peptide linker of thesingle-chain insulin analog is selected from the group consisting ofAGRGSGK (SEQ ID NO: 35), AGLGSGK (SEQ ID NO: 36), AGMGSGK (SEQ ID NO:37), ASWGSGK (SEQ ID NO: 38), TGLGSGQ (SEQ NO: 39), TGLGRGK (SEQ ID NO:40), TGLGSGK (SEQ ID NO: 41), HGLYSGK (SEQ ID NO: 42), KGLGSGQ (SEQ IDNO: 43), VGLMSGK (SEQ ID NO: 44), VGLSSGQ (SEQ ID NO: 45), VGLYSGK (SEQID NO: 46), VGLSSGK (SEQ ID NO: 47), VGMSSGK (SEQ ID NO: 48), VWSSSGK(SEQ ID NO: 49), VGSSSGK (SEQ ID NO: 50), VGMSSGK (SEQ ID NO: 51),TGLGSGR (SEQ ID NO: 52), TGLGKGQ (SEQ ID NO: 53), KGLSSGQ (SEQ ID NO:54), VKLSSGQ (SEQ ID NO: 55), VGLKSGQ (SEQ ID NO: 56), TGLGKGQ (SEQ IDNO: 57) and VGLSKGQ (SEQ ID NO: 58) wherein at least one lysine residuein the A-chain, in the B-chain or in the connecting peptide has beenchemically modified by acylation. In one embodiment the acylating groupcomprises a 1-5, 10-12 or 12-24 carbon chain.

In accordance with one embodiment the insulin prodrug analogs asdisclosed herein are further modified to link an additional compound tothe prodrug dipeptide moiety of the analog. In one embodiment the sidechain of an amino acid comprising the dipeptide prodrug element ispegylated, acylated or alkylated. In one embodiment the dipeptide isacylated with a group comprising a 1-5, 10-12 or 12-24 carbon chain. Inone embodiment the dipeptide is pegylated with a 40-80 KDa polyethyleneglycol chain. In one embodiment the dipeptide prodrug element ispegylated and the insulin peptide linked to the dipeptide is acylated,including, for example, acylation at the C-terminal lysine of the Bchain. In accordance with one embodiment a hydrophilic moiety or asequestering macromolecule is covalently linked to the R₂ side chain ofthe dipeptide comprising the general structure:

wherein R₂ is selected from the group consisting of (C₁-C₄ alkyl)OH,(C₁-C₄ alkyl)SH, and (C₁-C₄ alkyl)NH₂. In one embodiment R₂ is (C₃-C₄alkyl)NH₂. Sequestering macromolecules are known to those skilled in theart and include dextrans and large molecular weight polyethylene glycol(i.e., greater than or equal to 80 KDa) By linking the sequesteringmacromolecule to the dipeptide moiety, the prodrug will remainsequestered, while the active insulin peptide is slowly released basedon the kinetics of the cleavage of the dipeptide amide bond.

The present disclosure also encompasses other conjugates in whichinsulin prodrug analogs of the invention are linked, optionally viacovalent bonding, and optionally via a linker, to a conjugate. Linkagecan be accomplished by covalent chemical bonds, physical forces suchelectrostatic, hydrogen, ionic, van der Waals, or hydrophobic orhydrophilic interactions. A variety of non-covalent coupling systems maybe used, including biotin-avidin, ligand/receptor, enzyme/substrate,nucleic acid/nucleic acid binding protein, lipid/lipid binding protein,cellular adhesion molecule partners; or any binding partners orfragments thereof which have affinity for each other.

Exemplary conjugates include but are not limited to a heterologouspeptide or polypeptide (including for example, a plasma protein), atargeting agent, an immunoglobulin or portion thereof (e.g. variableregion, CDR, or Fc region), a diagnostic label such as a radioisotope,fluorophore or enzymatic label, a polymer including water solublepolymers, or other therapeutic or diagnostic agents. In one embodiment aconjugate is provided comprising an insulin prodrug analog of thepresent disclosure and a plasma protein, wherein the plasma protein isselected from the group consisting of albumin, transferin andfibrinogen. In one embodiment the plasma protein moiety of the conjugateis albumin or transferin. In one embodiment, the linker comprises achain of atoms from 1 to about 60, or 1 to 30 atoms or longer, 2 to 5atoms, 2 to 10 atoms, 5 to 10 atoms, or 10 to 20 atoms long. In oneembodiment, the chain atoms are all carbon atoms. In one embodiment, thechain atoms in the backbone of the linker are selected from the groupconsisting of C, O, N, and S. Chain atoms and linkers may be selectedaccording to their expected solubility (hydrophilicity) so as to providea more soluble conjugate. In one embodiment, the linker provides afunctional group that is subject to cleavage by an enzyme or othercatalyst or hydrolytic conditions found in the target tissue or organ orcell. In one embodiment, the length of the linker is long enough toreduce the potential for steric hindrance. If the linker is a covalentbond or a peptidyl bond and the conjugate is a polypeptide, the entireconjugate can be a fusion protein. Such peptidyl linkers may be anylength. Exemplary linkers are from about 1 to 50 amino acids in length,5 to 50, 3 to 5, 5 to 10, 5 to 15, or 10 to 30 amino acids in length.Such fusion proteins may alternatively be produced by recombinantgenetic engineering methods known to one of ordinary skill in the art.

Conjugates and Fusions

The present disclosure also encompasses other conjugates in which theinsulin analogs disclosed herein are linked, optionally via covalentbonding and optionally via a linker, to a conjugate moiety. Linkage canbe accomplished by covalent chemical bonds, physical forces suchelectrostatic, hydrogen, ionic, van der Waals, or hydrophobic orhydrophilic interactions. A variety of non-covalent coupling systems maybe used, including biotin-avidin, ligand/receptor, enzyme/substrate,nucleic acid/nucleic acid binding protein, lipid/lipid binding protein,cellular adhesion molecule partners; or any binding partners orfragments thereof which have affinity for each other.

The peptide can be linked to conjugate moieties via direct covalentlinkage by reacting targeted amino acid residues of the peptide with anorganic derivatizing agent that is capable of reacting with selectedside chains or the N- or C-terminal residues of these targeted aminoacids. Reactive groups on the peptide or conjugate include, e.g., analdehyde, amino, ester, thiol, α-haloacetyl, maleimido or hydrazinogroup. Derivatizing agents include, for example, maleimidobenzoylsulfosuccinimide ester (conjugation through cysteine residues),N-hydroxysuccinimide (through lysine residues), glutaraldehyde, succinicanhydride or other agents known in the art. Alternatively, the conjugatemoieties can be linked to the peptide indirectly through intermediatecarriers, such as polysaccharide or polypeptide carriers. Examples ofpolysaccharide carriers include aminodextran. Examples of suitablepolypeptide carriers include polylysine, polyglutamic acid, polyasparticacid, co-polymers thereof, and mixed polymers of these amino acids andothers, e.g., serines, to confer desirable solubility properties on theresultant loaded carrier.

Cysteinyl residues most commonly are reacted with α-haloacetates (andcorresponding amines), such as chloroacetic acid or chloroacetamide, togive carboxymethyl or carboxyamidomethyl derivatives. Cysteinyl residuesalso are derivatized by reaction with bromotrifluoroacetone,alpha-bromo-β-(5-imidozoyl)propionic acid, chloroacetyl phosphate,N-alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyldisulfide, p-chloromercuribenzoate, 2-chloromercuri-4-nitrophenol, orchloro-7-nitrobenzo-2-oxa-1,3-diazole.

Histidyl residues are derivatized by reaction with diethylpyrocarbonateat pH 5.5-7.0 because this agent is relatively specific for the histidylside chain. Para-bromophenacyl bromide also is useful; the reaction ispreferably performed in 0.1 M sodium cacodylate at pH 6.0.

Lysinyl and amino-terminal residues are reacted with succinic or othercarboxylic acid anhydrides. Derivatization with these agents has theeffect of reversing the charge of the lysinyl residues. Other suitablereagents for derivatizing alpha-amino-containing residues includeimidoesters such as methyl picolinimidate, pyridoxal phosphate,pyridoxal, chloroborohydride, trinitrobenzenesulfonic acid,O-methylisourea, 2,4-pentanedione, and transaminase-catalyzed reactionwith glyoxylate.

Arginyl residues are modified by reaction with one or severalconventional reagents, among them phenylglyoxal, 2,3-butanedione,1,2-cyclohexanedione, and ninhydrin. Derivatization of arginine residuesrequires that the reaction be performed in alkaline conditions becauseof the high pK_(a) of the guanidine functional group. Furthermore, thesereagents may react with the groups of lysine as well as the arginineepsilon-amino group.

The specific modification of tyrosyl residues may be made, withparticular interest in introducing spectral labels into tyrosyl residuesby reaction with aromatic diazonium compounds or tetranitromethane. Mostcommonly, N-acetylimidizole and tetranitromethane are used to formO-acetyl tyrosyl species and 3-nitro derivatives, respectively.

Carboxyl side groups (aspartyl or glutamyl) are selectively modified byreaction with carbodiimides (R—N═C═N—R′), where R and R′ are differentalkyl groups, such as 1-cyclohexyl-3-(2-morpholinyl-4-ethyl)carbodiimide or 1-ethyl-3-(4-azonia-4,4-dimethylpentyl) carbodiimide.Furthermore, aspartyl and glutamyl residues are converted to asparaginyland glutaminyl residues by reaction with ammonium ions.

Other modifications include hydroxylation of proline and lysine,phosphorylation of hydroxyl groups of seryl or threonyl residues,methylation of the alpha-amino groups of lysine, arginine, and histidineside chains (T. E. Creighton, Proteins: Structure and MolecularProperties, W.H. Freeman & Co., San Francisco, pp. 79-86 (1983)),deamidation of asparagines or glutamine, acetylation of the N-terminalamine, and/or amidation or esterification of the C-terminal carboxylicacid group.

Another type of covalent modification involves chemically orenzymatically coupling glycosides to the peptide. Sugar(s) may beattached to (a) arginine and histidine, (b) free carboxyl groups, (c)free sulfhydryl groups such as those of cysteine, (d) free hydroxylgroups such as those of serine, threonine, or hydroxyproline, (e)aromatic residues such as those of tyrosine, or tryptophan, or (f) theamide group of glutamine. These methods are described in WO87/05330published 11 Sep. 1987, and in Aplin and Wriston, CRC Crit. Rev.Biochem., pp. 259-306 (1981).

Exemplary conjugate moieties that can be linked to any of the insulinanalogs described herein include but are not limited to a heterologouspeptide or polypeptide (including for example, a plasma protein), atargeting agent, an immunoglobulin or portion thereof (e.g. variableregion, CDR, or Fc region), a diagnostic label such as a radioisotope,fluorophore or enzymatic label, a polymer including water solublepolymers, or other therapeutic or diagnostic agents. In one embodiment aconjugate is provided comprising an insulin analog disclosed herein anda plasma protein, wherein the plasma protein is selected form the groupconsisting of albumin, transferin, fibrinogen and globulins.

In one embodiment, the linker comprises a chain of atoms from 1 to about60, or 1 to 30 atoms or longer, 2 to 5 atoms, 2 to 10 atoms, 5 to 10atoms, or 10 to 20 atoms long. In one embodiment, the chain atoms areall carbon atoms. In one embodiment, the chain atoms in the backbone ofthe linker are selected from the group consisting of C, O, N, and S.Chain atoms and linkers may be selected according to their expectedsolubility (hydrophilicity) so as to provide a more soluble conjugate.In one embodiment, the linker provides a functional group that issubject to cleavage by an enzyme or other catalyst or hydrolyticconditions found in the target tissue or organ or cell. In oneembodiment, the length of the linker is long enough to reduce thepotential for steric hindrance. If the linker is a covalent bond or apeptidyl bond and the conjugate is a polypeptide, the entire conjugatecan be a fusion protein. Such peptidyl linkers may be any length.Exemplary linkers are from about 1 to 50 amino acids in length, 5 to 50,3 to 5, 5 to 10, 5 to 15, or 10 to 30 amino acids in length. Such fusionproteins may alternatively be produced by recombinant geneticengineering methods known to one of ordinary skill in the art.

As noted above, in one embodiment, the insulin analogs are conjugated,e.g., fused to an immunoglobulin or portion thereof (e.g. variableregion, CDR, or Fc region). Known types of immunoglobulins (Ig) includeIgG, IgA, IgE, IgD or IgM. The Fc region is a C-terminal region of an Igheavy chain, which is responsible for binding to Fc receptors that carryout activities such as recycling (which results in prolonged half-life),antibody dependent cell-mediated cytotoxicity (ADCC), and complementdependent cytotoxicity (CDC).

For example, according to some definitions the human IgG heavy chain Fcregion stretches from Cys226 to the C-terminus of the heavy chain. The“hinge region” generally extends from Glu216 to Pro230 of human IgG1(hinge regions of other IgG isotypes may be aligned with the IgG1sequence by aligning the cysteines involved in cysteine bonding). The Fcregion of an IgG includes two constant domains, CH2 and CH3. The CH2domain of a human IgG Fc region usually extends from amino acids 231 toamino acid 341. The CH3 domain of a human IgG Fc region usually extendsfrom amino acids 342 to 447. References made to amino acid numbering ofimmunoglobulins or immunoglobulin fragments, or regions, are all basedon Kabat et al. 1991, Sequences of Proteins of Immunological Interest,U.S. Department of Public Health, Bethesda, Md. In a relatedembodiments, the Fc region may comprise one or more native or modifiedconstant regions from an immunoglobulin heavy chain, other than CH1, forexample, the CH2 and CH3 regions of IgG and IgA, or the CH3 and CH4regions of IgE.

Suitable conjugate moieties include portions of immunoglobulin sequencethat include the FcRn binding site. FcRn, a salvage receptor, isresponsible for recycling immunoglobulins and returning them tocirculation in blood. The region of the Fc portion of IgG that binds tothe FcRn receptor has been described based on X-ray crystallography(Burmeister et al. 1994, Nature 372:379). The major contact area of theFc with the FcRn is near the junction of the CH2 and CH3 domains.Fc-FcRn contacts are all within a single Ig heavy chain. The majorcontact sites include amino acid residues 248, 250-257, 272, 285, 288,290-291, 308-311, and 314 of the CH2 domain and amino acid residues385-387, 428, and 433-436 of the CH3 domain.

Some conjugate moieties may or may not include FcγR binding site(s).FcγR are responsible for ADCC and CDC. Examples of positions within theFc region that make a direct contact with FcγR are amino acids 234-239(lower hinge region), amino acids 265-269 (B/C loop), amino acids297-299 (C′/E loop), and amino acids 327-332 (F/G) loop (Sondermann etal., Nature 406: 267-273, 2000). The lower hinge region of IgE has alsobeen implicated in the FcRI binding (Henry, et al., Biochemistry 36,15568-15578, 1997). Residues involved in IgA receptor binding aredescribed in Lewis et al., (J. Immunol. 175:6694-701, 2005). Amino acidresidues involved in IgE receptor binding are described in Sayers et al.(J Biol. Chem. 279(34):35320-5, 2004).

Amino acid modifications may be made to the Fc region of animmunoglobulin. Such variant Fc regions comprise at least one amino acidmodification in the CH3 domain of the Fc region (residues 342-447)and/or at least one amino acid modification in the CH2 domain of the Fcregion (residues 231-341). Mutations believed to impart an increasedaffinity for FcRn include T256A, T307A, E380A, and N434A (Shields et al.2001, J. Biol. Chem. 276:6591). Other mutations may reduce binding ofthe Fc region to FcγRI, FcγRIIA, FcγRIIB, and/or FcγRIIIA withoutsignificantly reducing affinity for FcRn. For example, substitution ofthe Asn at position 297 of the Fc region with Ala or another amino acidremoves a highly conserved N-glycosylation site and may result inreduced immunogenicity with concomitant prolonged half-life of the Fcregion, as well as reduced binding to FcγRs (Routledge et al. 1995,Transplantation 60:847; Friend et al. 1999, Transplantation 68:1632;Shields et al. 1995, J. Biol. Chem. 276:6591). Amino acid modificationsat positions 233-236 of IgG1 have been made that reduce binding to FcγRs(Ward and Ghetie 1995, Therapeutic Immunology 2:77 and Armour et al.1999, Eur. J. Immunol. 29:2613). Some exemplary amino acid substitutionsare described in U.S. Pat. Nos. 7,355,008 and 7,381,408, eachincorporated by reference herein in its entirety.

Acylation and Alkylation

In accordance with one embodiment, the insulin analogs disclosed hereinare modified to comprise an acyl group or alkyl group. Acylation oralkylation can increase the half-life of the insulin analogs incirculation. Acylation or alkylation can advantageously delay the onsetof action and/or extend the duration of action at the insulin and/orIGF-1 receptors and/or improve resistance to proteases such as DPP-IVand/or improve solubility. Insulin analogs may be acylated or alkylatedat the same amino acid position where a hydrophilic moiety is linked, orat a different amino acid position.

In one embodiment, the invention provides an insulin analog modified tocomprise an acyl group or alkyl group covalently linked to the aminoacid at a position corresponding to A10, B28, B29 of native insulin, orat the C-terminus or N-terminus of the A or B chain. The Insulin analogmay further comprise a spacer between the Insulin analog amino acid andthe acyl group or alkyl group. In one embodiment, the acyl group is afatty acid or bile acid, or salt thereof, e.g. a C4 to C30 fatty acid, aC8 to C24 fatty acid, cholic acid, a C4 to C30 alkyl, a C8 to C24 alkyl,or an alkyl comprising a steroid moiety of a bile acid. The spacer isany moiety with suitable reactive groups for attaching acyl or alkylgroups. In exemplary embodiments, the spacer comprises an amino acid, adipeptide, or a tripeptide, or a hydrophilic bifunctional spacer. In oneembodiment, the spacer is selected from the group consisting of: Trp,Glu, Asp, Cys and a spacer comprising NH₂(CH₂CH₂O)n(CH₂)mCOOH, wherein mis any integer from 1 to 6 and n is any integer from 2 to 12. Suchacylated or alkylated insulin peptides may also further comprise ahydrophilic moiety, optionally a polyethylene glycol. Any of theforegoing insulin analogs may comprise two acyl groups or two alkylgroups, or a combination thereof.

Acylation can be carried out at any positions within the insulin analog,provided that insulin analog insulin agonist activity is retained. Theacyl group can be covalently linked directly to an amino acid of theinsulin analog, or indirectly to an amino acid of the insulin analog viaa spacer, wherein the spacer is positioned between the amino acid of theinsulin peptide and the acyl group. In a specific aspect of theinvention, the insulin analog is modified to comprise an acyl group bydirect acylation of an amine, hydroxyl, or thiol of a side chain of anamino acid of the insulin peptide. In one embodiment, the insulin analogis directly acylated through the side chain amine, hydroxyl, or thiol ofan amino acid. In one embodiment, acylation is at a positioncorresponding to A10, B28, B29 of native insulin, or at the C-terminusor N-terminus of the A or B chain. In this regard, the acylated insulinanalog can comprise the amino acid sequence of SEQ ID NO: 9 and SEQ IDNO: 10, or a modified amino acid sequence thereof comprising one or moreof the amino acid modifications described herein, with at least one ofthe amino acids at a position corresponding to A10, B28, B29 of nativeinsulin, or at the C-terminus or N-terminus of the A or B chain modifiedto any amino acid comprising a side chain amine, hydroxyl, or thiol. Insome specific embodiments, the direct acylation of the insulin peptideoccurs through the side chain amine, hydroxyl, or thiol of the aminoacid at a position corresponding to A10, B28, B29 of native insulin. Inaccordance with one embodiment one of the amino acid side chains of thedipeptide element is acylated.

In one embodiment, the amino acid to be acylated is an amino acid ofFormula IV:

In some exemplary embodiments, the amino acid of Formula IV, is theamino acid wherein n is 4 (Lys) or n is 3 (Orn).

In other embodiments, the amino acid comprising a side chain hydroxyl isan amino acid of Formula V:

In some exemplary embodiments, the amino acid of Formula V is the aminoacid wherein n is 1 (Ser).

In yet other embodiments, the amino acid comprising a side chain thiolis an amino acid of Formula VI:

In some exemplary embodiments, the amino acid of Formula VI is the aminoacid wherein n is 1 (Cys).

In some exemplary embodiments, the insulin analog is modified tocomprise an acyl group by acylation of an amine, hydroxyl, or thiol of aspacer, which spacer is attached to a side chain of an amino acid atposition A10, B28 or B29 (according to the amino acid numbering of wildtype insulin). The amino acid to which the spacer is attached can be anyamino acid comprising a moiety which permits linkage to the spacer. Forexample, an amino acid comprising a side chain NH2, —OH, or —COOH (e.g.,Lys, Orn, Ser, Asp, or Glu) is suitable. In one embodiment, the spaceris an amino acid comprising a side chain amine, hydroxyl, or thiol, or adipeptide or tripeptide comprising an amino acid comprising a side chainamine, hydroxyl, or thiol.

When acylation occurs through an amine group of a spacer the acylationcan occur through the alpha amine of the amino acid or a side chainamine. In the instance in which the alpha amine is acylated, the spaceramino acid can be any amino acid. For example, the spacer amino acid canbe a hydrophobic amino acid, e.g., Gly, Ala, Val, Leu, Ile, Trp, Met,Phe, Tyr. Alternatively, the spacer amino acid can be an acidic residue,e.g., Asp and Glu. In the instance in which the side chain amine of thespacer amino acid is acylated, the spacer amino acid is an amino acidcomprising a side chain amine, e.g., an amino acid of Formula I (e.g.,Lys or Orn). In this instance, it is possible for both the alpha amineand the side chain amine of the spacer amino acid to be acylated, suchthat the insulin peptide is diacylated. The present disclosure furthercontemplates diacylated insulin analogs.

When acylation occurs through a hydroxyl group of a spacer, the aminoacid or one of the amino acids of the dipeptide or tripeptide can be anamino acid of Formula II. In a specific exemplary embodiment, the aminoacid is Ser.

When acylation occurs through a thiol group of a spacer, the amino acidor one of the amino acids of the dipeptide or tripeptide can be an aminoacid of Formula III. In a specific exemplary embodiment, the amino acidis Cys.

In one embodiment, the spacer comprises a hydrophilic bifunctionalspacer. In a specific embodiment, the spacer comprises an aminopoly(alkyloxy)carboxylate. In this regard, the spacer can comprise, forexample, NH₂(CH₂CH₂O)_(n)(CH₂)_(m)COOH, wherein m is any integer from 1to 6 and n is any integer from 2 to 12, such as, e.g.,8-amino-3,6-dioxaoctanoic acid, which is commercially available fromPeptides International, Inc. (Louisville, Ky.).

Suitable methods of peptide acylation via amines, hydroxyls, and thiolsare known in the art. See, for example, Miller, Biochem Biophys ResCommun 218: 377-382 (1996); Shimohigashi and Stammer, Int J Pept ProteinRes 19: 54-62 (1982); and Previero et al., Biochim Biophys Acta 263:7-13 (1972) (for methods of acylating through a hydroxyl); and San andSilvius, J Pept Res 66: 169-180 (2005) (for methods of acylating througha thiol); Bioconjugate Chem. “Chemical Modifications of Proteins:History and Applications” pages 1, 2-12 (1990); Hashimoto et al.,Pharmacuetical Res. “Synthesis of Palmitoyl Derivatives of Insulin andtheir Biological Activity” Vol. 6, No: 2 pp. 171-176 (1989).

The acyl group of the acylated insulin peptide can be of any size, e.g.,any length carbon chain, and can be linear or branched. In some specificembodiments of the invention, the acyl group is a C4 to C30 fatty acid.For example, the acyl group can be any of a C4 fatty acid, C6 fattyacid, C8 fatty acid, C10 fatty acid, C12 fatty acid, C14 fatty acid, C16fatty acid, C18 fatty acid, C20 fatty acid, C22 fatty acid, C24 fattyacid, C26 fatty acid, C28 fatty acid, or a C30 fatty acid. In oneembodiment, the acyl group is a C8 to C20 fatty acid, e.g., a C14 fattyacid or a C16 fatty acid.

In an alternative embodiment, the acyl group is a bile acid. The bileacid can be any suitable bile acid, including, but not limited to,cholic acid, chenodeoxycholic acid, deoxycholic acid, lithocholic acid,taurocholic acid, glycocholic acid, and cholesterol acid.

In a specific embodiment, the insulin analog comprises a cholesterolacid, which is linked to a Lys residue of the insulin analog through analkylated des-amino Cys spacer, i.e., an alkylated 3-mercaptopropionicacid spacer. The alkylated des-amino Cys spacer can be, for example, ades-amino-Cys spacer comprising a dodecaethylene glycol moiety. In oneembodiment, the insulin analog comprises the structure:

The acylated insulin analogs described herein can be further modified tocomprise a hydrophilic moiety. In some specific embodiments thehydrophilic moiety can comprise a polyethylene glycol (PEG) chain. Theincorporation of a hydrophilic moiety can be accomplished through anysuitable means, such as any of the methods described herein.

Alternatively, the acylated insulin peptide can comprise a spacer,wherein the spacer is both acylated and modified to comprise thehydrophilic moiety. Nonlimiting examples of suitable spacers include aspacer comprising one or more amino acids selected from the groupconsisting of Cys, Lys, Orn, homo-Cys, and Ac-Phe.

In accordance with one embodiment, the insulin analog is modified tocomprise an alkyl group which is attached to the insulin analog via anester, ether, thioether, amide, or alkyl amine linkage for purposes ofprolonging half-life in circulation and/or delaying the onset of and/orextending the duration of action and/or improving resistance toproteases such as DPP-IV.

The alkyl group of the alkylated insulin peptide can be of any size,e.g., any length carbon chain, and can be linear or branched. In oneembodiment of the invention, the alkyl group is a C1 to C30 alkyl. Forexample, the alkyl group can be any of a C1 alkyl, C2 alkyl, C3 alkyl,C4 alkyl, C6 alkyl, C8 alkyl, C10 alkyl, C12 alkyl, C14 alkyl, C16alkyl, C18 alkyl, C20 alkyl, C22 alkyl, C24 alkyl, C26 alkyl, C28 alkyl,or a C30 alkyl. In one embodiment, the alkyl group is a C8 to C20 alkyl,e.g., a C14 alkyl or a C16 alkyl.

In some specific embodiments, the alkyl group comprises a steroid moietyof a bile acid, e.g., cholic acid, chenodeoxycholic acid, deoxycholicacid, lithocholic acid, taurocholic acid, glycocholic acid, andcholesterol acid.

In accordance with one embodiment a pharmaceutical composition isprovided comprising any of the novel insulin prodrug analogs disclosedherein, preferably at a purity level of at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98% or 99%, and a pharmaceutically acceptablediluent, carrier or excipient. Such compositions may contain an A19insulin analog as disclosed herein at a concentration of at least 0.5mg/ml, 1 mg/ml, 2 mg/ml, 3 mg/ml, 4 mg/ml, 5 mg/ml, 6 mg/ml, 7 mg/ml, 8mg/ml, 9 mg/ml, 10 mg/ml, 11 mg/ml, 12 mg/ml, 13 mg/ml, 14 mg/ml, 15mg/ml, 16 mg/ml, 17 mg/ml, 18 mg/ml, 19 mg/ml, 20 mg/ml, 21 mg/ml, 22mg/ml, 23 mg/ml, 24 mg/ml, 25 mg/ml or higher. In one embodiment thepharmaceutical compositions comprise aqueous solutions that aresterilized and optionally stored contained within various packagecontainers. In other embodiments the pharmaceutical compositionscomprise a lyophilized powder. The pharmaceutical compositions can befurther packaged as part of a kit that includes a disposable device foradministering the composition to a patient. The containers or kits maybe labeled for storage at ambient room temperature or at refrigeratedtemperature.

In one embodiment, a composition is provided comprising a mixture of afirst and second insulin prodrug analog, wherein the first and secondinsulin prodrug analogs differ from one another based on the structureof the prodrug element. More particularly, the first insulin prodruganalog may comprise a dipeptide prodrug element that has a half lifesubstantially different from the dipeptide prodrug element of the secondinsulin prodrug analog. Accordingly, selection of different combinationsof substituents on the dipeptide element will allow for the preparationof compositions that comprise a mixture of insulin prodrug analogs thatare activated in a controlled manner over a desired time frame and atspecific time intervals. For example, the compositions can be formulatedto release active insulin at mealtimes followed by a subsequentactivation of insulin during nighttime with suitable dosages beingreleased based on time of activation. In another embodiment thepharmaceutical composition comprises a mixture of an insulin prodruganalog disclosed herein and native insulin, or a known bioactivederivative of insulin.

The disclosed insulin prodrug analogs are believed to be suitable forany use that has previously been described for insulin peptides.Accordingly, the insulin prodrug analogs described herein can be used totreat hyperglycemia, or treat other metabolic diseases that result fromhigh blood glucose levels. Accordingly, the present inventionencompasses pharmaceutical compositions comprising an insulin prodruganalog of the present disclosure, and a pharmaceutically acceptablecarrier for use in treating a patient suffering from high blood glucoselevels. In accordance with one embodiment the patient to be treatedusing the insulin prodrug analogs disclosed herein is a domesticatedanimal, and in another embodiment the patient to be treated is a human.

One method of treating hyperglycemia in accordance with the presentdisclosure comprises the steps of administering the presently disclosedinsulin prodrug analog to a patient using any standard route ofadministration, including parenterally, such as intravenously,intraperitoneally, subcutaneously or intramuscularly, intrathecally,transdermally, rectally, orally, nasally or by inhalation. In oneembodiment the composition is administered subcutaneously orintramuscularly. In one embodiment, the composition is administeredparenterally and the insulin prodrug analog composition is prepackagedin a syringe.

The insulin prodrug analogs of the invention may be administered aloneor in combination with other anti-diabetic agents. Anti-diabetic agentsknown in the art or under investigation include native insulin, nativeglucagon and functional derivatives thereof, sulfonylureas, such astolbutamide (Orinase), acetohexamide (Dymelor), tolazamide (Tolinase),chlorpropamide (Diabinese), glipizide (Glucotrol), glyburide (Diabeta,Micronase, Glynase), glimepiride (Amaryl), or gliclazide (Diamicron);meglitinides, such as repaglinide (Prandin) or nateglinide (Starlix);biguanides such as metformin (Glucophage) or phenformin;thiazolidinediones such as rosiglitazone (Avandia), pioglitazone(Actos), or troglitazone (Rezulin), or other PPARγ inhibitors; alphaglucosidase inhibitors that inhibit carbohydrate digestion, such asmiglitol (Glyset), acarbose (Precose/Glucobay); exenatide (Byetta) orpramlintide; Dipeptidyl peptidase-4 (DPP-4) inhibitors such asvildagliptin or sitagliptin; SGLT (sodium-dependent glucosetransporter 1) inhibitors; or FBPase (fructose 1,6-bisphosphatase)inhibitors.

Pharmaceutical compositions comprising the insulin prodrug analogsdisclosed herein can be formulated and administered to patients usingstandard pharmaceutically acceptable carriers and routes ofadministration known to those skilled in the art. Accordingly, thepresent disclosure also encompasses pharmaceutical compositionscomprising one or more of the insulin prodrug analogs disclosed herein,or a pharmaceutically acceptable salt thereof, in combination with apharmaceutically acceptable carrier. In one embodiment thepharmaceutical composition comprises a 1 mg/ml concentration of theinsulin prodrug analog at pH of about 4.0 to about 7.0 in a phosphatebuffer system. The pharmaceutical compositions may comprise the insulinprodrug analog as the sole pharmaceutically active component, or theinsulin prodrug analog can be combined with one or more additionalactive agents. In accordance with one embodiment a pharmaceuticalcomposition is provided comprising one of the insulin prodrug analogsdisclosed herein, preferably sterile and preferably at a purity level ofat least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, and apharmaceutically acceptable diluent, carrier or excipient. Suchcompositions may contain an insulin prodrug analog wherein the resultingactive peptide is present at a concentration of at least 0.5 mg/ml, 1mg/ml, 2 mg/ml, 3 mg/ml, 4 mg/ml, 5 mg/ml, 6 mg/ml, 7 mg/ml, 8 mg/ml, 9mg/ml, 10 mg/ml, 11 mg/ml, 12 mg/ml, 13 mg/ml, 14 mg/ml, 15 mg/ml, 16mg/ml, 17 mg/ml, 18 mg/ml, 19 mg/ml, 20 mg/ml, 21 mg/ml, 22 mg/ml, 23mg/ml, 24 mg/ml, 25 mg/ml or higher. In one embodiment thepharmaceutical compositions comprise aqueous solutions that aresterilized and optionally stored within various containers. Thecompounds of the present invention can be used in accordance with oneembodiment to prepare pre-formulated solutions ready for injection. Inother embodiments the pharmaceutical compositions comprise a lyophilizedpowder. The pharmaceutical compositions can be further packaged as partof a kit that includes a disposable device for administering thecomposition to a patient. The containers or kits may be labeled forstorage at ambient room temperature or at refrigerated temperature.

All therapeutic methods, pharmaceutical compositions, kits and othersimilar embodiments described herein contemplate that insulin prodruganalogs include all pharmaceutically acceptable salts thereof.

In one embodiment the kit is provided with a device for administeringthe insulin prodrug analog composition to a patient. The kit may furtherinclude a variety of containers, e.g., vials, tubes, bottles, and thelike. Preferably, the kits will also include instructions for use. Inaccordance with one embodiment the device of the kit is an aerosoldispensing device, wherein the composition is prepackaged within theaerosol device. In another embodiment the kit comprises a syringe and aneedle, and in one embodiment the insulin analog composition isprepackaged within the syringe.

The compounds of this invention may be prepared by standard syntheticmethods, recombinant DNA techniques, or any other methods of preparingpeptides and fusion proteins. Although certain non-natural amino acidscannot be expressed by standard recombinant DNA techniques, techniquesfor their preparation are known in the art. Compounds of this inventionthat encompass non-peptide portions may be synthesized by standardorganic chemistry reactions, in addition to standard peptide chemistryreactions when applicable.

EXAMPLE 1 Synthesis of Insulin A & B Chains

Insulin A & B chains were synthesized on 4-methylbenzhyryl amine (MBHA)resin or 4-Hydroxymethyl-phenylacetamidomethyl (PAM) resin using Bocchemistry. The peptides were cleaved from the resin using HF/p-cresol95:5 for 1 hour at 0° C. Following HF removal and ether precipitation,peptides were dissolved into 50% aqueous acetic acid and lyophilized.Alternatively, peptides were synthesized using Fmoc chemistry. Thepeptides were cleaved from the resin using Trifluoroacetic acid(TFA)/Triisopropylsilane (TIS)/H₂O (95:2.5:2.5), for 2 hour at roomtemperature. The peptide was precipitated through the addition of anexcessive amount of diethyl ether and the pellet solubilized in aqueousacidic buffer. The quality of peptides were monitored by RP-HPLC andconfirmed by Mass Spectrometry (ESI or MALDI).

Insulin A chains were synthesized with a single free cysteine at aminoacid 7 and all other cysteines protected as acetamidomethylA-(SH)⁷(Acm)^(6,11,20). Insulin B chains were synthesized with a singlefree cysteine at position 7 and the other cysteine protected asacetamidomethyl B-(SH)⁷(Acm)¹⁹. The crude peptides were purified byconventional RP-HPLC.

The synthesized A and B chains were linked to one another through theirnative disulfide bond linkage in accordance with the general procedureoutlined in FIG. 1. The respective B chain was activated to theCys⁷-Npys derivative through dissolution in DMF or DMSO and reacted with2,2′-Dithiobis(5-nitropyridine) (Npys) at a 1:1 molar ratio, at roomtemperature. The activation was monitored by RP-HPLC and the product wasconfirmed by ESI-MS.

The first B7-A7 disulfide bond was formed by dissolution of therespective A-(SH)⁷(Acm)^(6,11,20) and B-(Npys)⁷(Acm)¹⁹ at 1:1 molarratio to a total peptide concentration of 10 mg/ml. When the chaincombination reaction was complete the mixture was diluted to aconcentration of 50% aqueous acetic acid. The last two disulfide bondswere formed simultaneously through the addition of iodine. A 40 foldmolar excess of iodine was added to the solution and the mixture wasstirred at room temperature for an additional hour. The reaction wasterminated by the addition of an aqueous ascorbic acid solution. Themixture was purified by RP-HPLC and the final compound was confirmed byMALDI-MS. As shown in FIG. 2 and the data in Table 1, the syntheticinsulin prepared in accordance with this procedure compares well withpurified insulin for insulin receptor binding.

Insulin peptides comprising a modified amino acid (such as 4-aminophenylalanine at position A19) can also be synthesized in vivo using asystem that allows for incorporation of non-coded amino acids intoproteins, including for example, the system taught in U.S. Pat. Nos.7,045,337 and 7,083,970.

TABLE 1 Activity of synthesized insulin relative to native insulinInsulin Standard A7-B7 Insulin AVER. STDEV AVER. STDEV IC₅₀(nM) 0.240.07 0.13 0.08 % of Insulin Activity 100 176.9

EXAMPLE 2 Pegylation of Amine Groups (N-Terminus and Lysine) byReductive Alkylation

a. Synthesis

Insulin (or an insulin analog), mPEG20k-Aldyhyde, and NaBH₃CN, in amolar ratio of 1:2:30, were dissolved in acetic acid buffer at a pH of4.1-4.4. The reaction solution was composed of 0.1 N NaCl, 0.2 N aceticacid and 0.1 N Na₂CO₃. The insulin peptide concentration wasapproximately 0.5 mg/ml. The reaction occurs over six hours at roomtemperature. The degree of reaction was monitored by RP-HPLC and theyield of the reaction was approximately 50%.

b. Purification

The reaction mixture was diluted 2-5 fold with 0.1% TFA and applied to apreparative RP-HPLC column. HPLC condition: C4 column; flow rate 10ml/min; A buffer 10% ACN and 0.1% TFA in water; B buffer 0.1% TFA inACN; A linear gradient B % from 0-40% (0-80 min); PEG-insulin oranalogues was eluted at approximately 35% buffer B. The desiredcompounds were verified by MALDI-TOF, following chemical modificationthrough sulftolysis or trypsin degradation.

Pegylation of Amine Groups (N-Terminus and Lysine) byN-Hydroxysuccinimide Acylation.

a. Synthesis

Insulin (or an insulin analog) along with mPEG20k-NHS were dissolved in0.1 N Bicine buffer (pH 8.0) at a molar ratio of 1:1. The insulinpeptide concentration was approximately 0.5 mg/ml. Reaction progress wasmonitored by HPLC. The yield of the reaction is approximately 90% after2 hours at room temperature.

b. Purification

The reaction mixture was diluted 2-5 fold and loaded to RP-HPLC. HPLCcondition: C4 column; flow rate 10 ml/min; A buffer 10% ACN and 0.1% TFAin water; B buffer 0.1% TFA in ACN; A linear gradient B % from 0-40%(0-80 min); PEG-insulin or analogues was collected at approximately 35%B. The desired compounds were verified by MAIDI-TOF, following chemicalmodification through sulftolysis or trypsin degradation.

Reductive Aminated Pegylation of Acetyl Group on the Aromatic Ring ofthe Phenylalanine

a. Synthesis

Insulin (or an insulin analogue), mPEG20k-Hydrazide, and NaBH₃CN in amolar ratio of 1:2:20 were dissolved in acetic acid buffer (pH of 4.1 to4.4). The reaction solution was composed of 0.1 N NaCl, 0.2 N aceticacid and 0.1 N Na₂CO₃. Insulin or insulin analogue concentration wasapproximately 0.5 mg/ml. at room temperature for 24 h. The reactionprocess was monitored by HPLC. The conversion of the reaction wasapproximately 50%. (calculated by HPLC)

b. Purification

The reaction mixture was diluted 2-5 fold and loaded to RP-HPLC. HPLCcondition: C4 column; flow rate 10 ml/min; A buffer 10% ACN and 0.1% TFAin water; B buffer 0.1% TFA in ACN; A linear gradient B % from 0-40%(0-80 min); PEG-insulin, or the PEG-insulin analogue was collected atapproximately 35% B. The desired compounds were verified by MAIDI-TOF,following chemical modification through sulftolysis or trypsindegradation.

EXAMPLE 3 Insulin Receptor Binding Assay

The affinity of each peptide for the insulin or IGF-1 receptor wasmeasured in a competition binding assay utilizing scintillationproximity technology. Serial 3-fold dilutions of the peptides were madein Tris-Cl buffer (0.05 M Tris-HCl, pH 7.5, 0.15 M NaCl, 0.1% w/v bovineserum albumin) and mixed in 96 well plates (Corning Inc., Acton, Mass.)with 0.05 nM (3-[125I]-iodotyrosyl) A TyrA14 insulin or(3-[125I]-iodotyrosyl) IGF-1 (Amersham Biosciences, Piscataway, N.J.).An aliquot of 1-6 micrograms of plasma membrane fragments prepared fromcells over-expressing the human insulin or IGF-1 receptors were presentin each well and 0.25 mg/well polyethylene imine-treated wheat germagglutinin type A scintillation proximity assay beads (AmershamBiosciences, Piscataway, N.J.) were added. After five minutes of shakingat 800 rpm the plate was incubated for 12 h at room temperature andradioactivity was measured with MicroBeta1450 liquid scintillationcounter (Perkin-Elmer, Wellesley, Mass.). Non-specifically bound (NSB)radioactivity was measured in the wells with a four-fold concentrationexcess of “cold” native ligand than the highest concentration in testsamples. Total bound radioactivity was detected in the wells with nocompetitor. Percent specific binding was calculated as following: %Specific Binding=(Bound-NSB/Total bound-NSB)×100. IC50 values weredetermined by using Origin software (OriginLab, Northampton, Mass.).

EXAMPLE 4 Insulin Receptor Phosphorylation Assay

To measure receptor phosphorylation of insulin or insulin analog,receptor transfected HEK293 cells were plated in 96 well tissue cultureplates (Costar #3596, Cambridge, Mass.) and cultured in Dulbecco'smodified Eagle medium (DMEM) supplemented with 100 IU/ml penicillin, 100μg/ml streptomycin, 10 mM HEPES and 0.25% bovine growth serum (HyCloneSH30541, Logan, Utah) for 16-20 hrs at 37° C., 5% CO₂ and 90% humidity.Serial dilutions of insulin or insulin analogs were prepared in DMEMsupplemented with 0.5% bovine serum albumin (Roche Applied Science#100350, Indianapolis, Ind.) and added to the wells with adhered cells.After 15 min incubation at 37° C. in humidified atmosphere with 5% CO₂the cells were fixed with 5% paraformaldehyde for 20 min at roomtemperature, washed twice with phosphate buffered saline pH 7.4 andblocked with 2% bovine serum albumin in PBS for 1 hr. The plate was thenwashed three times and filled with horseradish peroxidase-conjugatedantibody against phosphotyrosine (Upstate biotechnology #16-105,Temecula, Calif.) reconstituted in PBS with 2% bovine serum albumin permanufacturer's recommendation. After 3 hrs incubation at roomtemperature the plate was washed 4 times and 0.1 ml of TMB singlesolution substrate (Invitrogen, #00-2023, Carlbad, Calif.) was added toeach well. Color development was stopped 5 min later by adding 0.05 ml 1N HCl. Absorbance at 450 nm was measured on Titertek Multiscan MCC340(ThermoFisher, Pittsburgh, Pa.). Absorbance vs. peptide concentrationdose response curves were plotted and EC₅₀ values were determined byusing Origin software (OriginLab, Northampton, Mass.).

EXAMPLE 5 Determination of Rate of Model Dipeptide Cleavage (in PBS)

A specific hexapeptide (HSRGTF-NH₂; SEQ ID NO: 59) was used as a modelpeptide upon which the rate of cleavage of dipeptide N-terminalextensions could be studied. The dipeptide-extended model peptides wereprepared Boc-protected sarcosine and lysine were successively added tothe model peptide-bound resin to produce peptide A (Lys-Sar-HSRGTF-NH₂;SEQ ID NO: 60). Peptide A was cleaved by HF and purified by preparativeHPLC.

The rate of cleavage was determined for the respective propeptides. Theconcentrations of the propeptides and the model parent peptide weredetermined by their respective peak areas. The first order dissociationrate constants of the prodrugs were determined by plotting the logarithmof the concentration of the prodrug at various time intervals. The slopeof this plot provides the rate constant ‘k’. The half lives for cleavageof the various prodrugs were calculated by using the formulat_(1/2)=0.693/k. The half life of the Lys-Sar extension to this modelpeptide HSRGTF-NH₂ (SEQ ID NO: 59) was determined to be 14.0 h.

EXAMPLE 6 Rate of Dipeptide Cleavage Half Time in Plasma as Determinedwith an all d-Isoform Model Peptide

An additional model hexapeptide (dHdTdRGdTdF-NH₂ SEQ ID NO: 63) was usedto determine the rate of dipeptide cleavage in plasma. The d-isomer ofeach amino acid was used to prevent enzymatic cleavage of the modelpeptide, with the exception of the prodrug extension. This modeld-isomer hexapeptide was synthesized in an analogous fashion to the1-isomer. The sarcosine and lysine were successively added to theN-terminus as reported previously for peptide A to prepare peptide B(dLys-dSar-dHdTdRGdTdF-NH₂ SEQ ID NO: 64)

The rate of cleavage was determined for the respective propeptides. Theconcentrations of the propeptides and the model parent peptide weredetermined by their respective peak areas. The first order dissociationrate constants of the prodrugs were determined by plotting the logarithmof the concentration of the prodrug at various time intervals. The slopeof this plot provides the rate constant ‘k’. The half life of theLys-Sar extension to this model peptide dHdTdRGdTdF-NH₂ (SEQ ID NO: 63)was determined to be 18.6 h.

EXAMPLE 7

The rate of cleavage for additional dipeptides linked to the modelhexapeptide (HSRGTF-NH₂; SEQ ID NO: 59) were determined using theprocedures described in Example 5. The results generated in theseexperiments are presented in Tables 2 and 3.

TABLE 2 Cleavage of the Dipeptides O-U that are linked to the side chainof an N-terminal para-amino-Phe from the Model Hexapeptide (HSRGTF-NH₂;SEQ ID NO: 59) in PBS

Compounds U (amino acid) O (amino acid) t_(1/2) 1 F P 58 h 2 Hydroxyl-FP 327 h 3 d-F P 20 h 4 d-F d-P 39 h 5 G P 72 h 6 Hydroxyl-G P 603 h 7 LP 62 h 8 tert-L P 200 h 9 S P 34 h 10 P P 97 h 11 K P 33 h 12 dK P 11 h13 E P 85 h 14 Sar P ≈1000 h 15 Aib P 69 min 16 Hydroxyl-Aib P 33 h 17cyclohexane P 6 min 18 G G No cleavage 19 Hydroxyl-G G No cleavage 20 SN-Methyl-Gly 4.3 h 21 K N-Methyl-Gly 5.2 h 22 Aib N-Methyl-Gly 7.1 min23 Hydroxyl-Aib N-Methyl-Gly 1.0 h

TABLE 3 Cleavage of the Dipeptides U-O linked to histidine (or histidinederivative) at position 1 (X) from the Model Hexapeptide (XSRGTF-NH₂;SEQ ID NO: 59) in PBS NH₂-U-O-XSRGTF-NH₂ Cmp U (amino acid) O (aminoacid) X (amino acid) t_(1/2) 1 F P H No cleavage 2 Hydroxyl-F P H Nocleavage 3 G P H No cleavage 4 Hydroxyl-G P H No cleavage 5 A P H Nocleavage 6 C P H No cleavage 7 S P H No cleavage 8 P P H No cleavage 9 KP H No cleavage 10 E P H No cleavage 11 Dehydro V P H No cleavage 12 Pd-P H No cleavage 13 d-P P H No cleavage 14 Aib P H 32 h 15 Aib d-P H 20h 16 Aib P d-H 16 h 17 Cyclohexyl- P H 5 h 18 Cyclopropyl- P H 10 h 19N—Me-Aib P H >500 h 20 α, α-diethyl-Gly P H 46 h 21 Hydroxyl-Aib P H 6122 Aib P A 58 23 Aib P N-Methyl-His 30 h 24 Aib N-Methyl-Gly H 49 min 25Aib N-Hexyl-Gly H 10 min 26 Aib Azetidine-2- H >500 h carboxylic acid 27G N-Methyl-Gly H 104 h 28 Hydroxyl-G N-Methyl-Gly H 149 h 29 GN-Hexyl-Gly H 70 h 30 dK N-Methyl-Gly H 27 h 31 dK N-Methyl-Ala H 14 h32 dK N-Methyl-Phe H 57 h 33 K N-Methyl-Gly H 14 h 34 F N-Methyl-Gly H29 h 35 S N-Methyl-Gly H 17 h 36 P N-Methyl-Gly H 181 h

EXAMPLE 8 Identification of an Insulin Analog with Structure Suitablefor Prodrug Construction

Position 19 of the A chain is known to be an important site for insulinactivity. Modification at this site to allow the attachment of a prodrugelement is therefore desirable. Specific analogs of insulin at A19 havebeen synthesized and characterized for their activity at the insulinreceptors. Two highly active structural analogs have been identified atA19, wherein comparable structural changes at a second active sitearomatic residue (B24) were not successful in identification ofsimilarly full activity insulin analogs.

Tables 4 and 5 illustrate the high structural conservation at positionA19 for full activity at the insulin receptor (receptor bindingdetermined using the assay described in Example 3). Table 4 demonstratesthat only two insulin analogs with modifications at A19 have receptorbinding activities similar to native insulin. For the 4-amino insulinanalog, data from three separate experiments is provided. The columnlabeled “Activity (in test)” compares the percent binding of the insulinanalog relative to native insulin for two separate experiments conductedsimultaneously. The column labeled “Activity (0.60 nM)” is the relativepercent binding of the insulin analog relative to the historical averagevalue obtained for insulin binding using this assay. Under eitheranalysis, two A19 insulin analogs (4-amino phenylalanine and 4-methoxyphenylalanine) demonstrate receptor binding approximately equivalent tonative insulin. FIG. 3 represents a graph demonstrating the respectivespecific binding of native insulin and the A19 insulin analog to theinsulin receptor. Table 5 presents data showing that the two A19 insulinanalogs (4-amino and 4-methoxy) that demonstrate equivalent bindingactivities as native insulin also demonstrate equivalent activity at theinsulin receptor (receptor activity determined using the assay describedin Example 4).

TABLE 4 Insulin Receptor Binding Activity of A19 Insulin Analogs InsulinReceptor % native ligand % native ligand Activity Analogue IC₅₀ STDevActivity (in test) (0.60 nM) 4-OH (native insulin) 0.64 0.15 100.0 100.04-COCH₃ 31.9 9.47 0.6 1.9 4-NH₂ 0.31 0.12 203.0 193.5 0.83 0.15 103.072.3 0.8 0.1 94.0 75.0 4-NO₂ 215.7 108.01 0.3 1.3 3,4,5-3F 123.29 31.100.5 0.5 4-OCH₃ 0.5 0.50 173.0 120.0 3-OCH₃ 4.74 1.09 28.0 12.7 5.16 3.8818.0 11.6 4-OH, 3,5-2Br 1807.17 849.72 0.0 0.0 4-OH, 3,5-2NO₂ 2346.2338.93 0.0 0.0

TABLE 5 Insulin Receptor Phosphorylation Activity of A19 Insulin AnalogsInsulin Receptor % native ligand Analogue EC₅₀ STDev Activity (in test)4-OH (native insulin) 1.22 0.4 100.0 4-NH₂ 0.31 0.14 393.5 4-OCH₃ 0.940.34 129.8

EXAMPLE 9 Insulin Like Growth Factor (IGF) Analog

Typical purification schemes for isolating the insulin A-chain use anNH₄HCO₃ buffer (pH=7.8). Under these conditions the dipeptide prodrugelement is rapidly cleaved from the A-chain. To simplify purification ofprodrugs to investigate their activities at the insulin receptor,applicants conducted such studies using an IGF analog that demonstratessimilar activity at the insulin receptor as native insulin. Moreparticularly, the IGF analog (IGF1 (Y^(B16)L^(B17)) comprises the nativeIGF A and B chain (SEQ ID NO: 61 and SEQ ID NO: 62, respectively),wherein the native glutamine and phenylalanine at positions 15 and 16 ofthe native IGF B-chain (corresponding to positions 16 and 17 of nativeinsulin B-chain, respectively) have been replaced with tyrosine andleucine residues, respectively. As shown in FIG. 4 and Table 6 below thebinding activities of IGF1 (Y^(B16)L^(B17)) demonstrate the compound isa highly potent insulin analog.

TABLE 6 Insulin Standard IGF1(Y^(B16)L^(B17)) AVER. STDEV AVER. STDEVIC₅₀(nM) 1.32 0.19 0.51 0.18 % of Insulin Activity 100 262

EXAMPLE 10 IGF Prodrug Derivatives

Based on the activity of the A19 insulin analog (see Example 5), asimilar modification was made to the IGF1 A:B(Y^(B16)L^(B17)) analog andits ability to bind and stimulate insulin receptor activity wasinvestigated. FIG. 5 provides the general synthetic scheme for preparingIGF1 A:B(Y^(B16)L^(B17)) wherein the native tyrosine is replace with a4-amino phenylalanine [IGF1 A:B(Y^(B16)L^(B17))(p-NH₂—F)^(A19)amide] aswell as the preparation of its dipeptide extended derivative [IGF1A:B(Y^(B16)L^(B17))^(A19)-AiBAla amide], wherein a dipeptide comprisingAiB and Ala are linked to the peptide through an amide linkage to theA19 4-amino phenylalanine. As shown in FIG. 6 and Table 7, the IGFanalog, IGF1 (Y^(B16)L^(B17)) A(p-NH₂—F)¹⁹ specifically binds to theinsulin receptor wherein the dipeptide extended derivative of thatanalog fails to specifically bind the insulin receptor. Note thedipeptide extension lacks the proper structure to allow for spontaneouscleavage of the dipeptide (absence of an N-alkylated amino acid at thesecond position of the dipeptide) and therefore there is no restorationof insulin receptor binding.

TABLE 7 IGF1(Y^(B16)L^(B17)) IGF1(Y^(B16)L^(B17)) Insulin Standard(p-NH₂—F)^(A19)amide (AiBAla)^(A19)amide AVER. STDEV AVER. STDEV. AVER.STDEV IC₅₀(nM) 0.24 0.07 1.08 .075 No Activity % of 100 22 InsulinActivity

A further prodrug derivative of an IGF^(B16B17) derivative peptide wasprepared wherein the dipeptide prodrug element (alanine-proline) waslinked via an amide bond to the amino terminus of the A chain(IGF1(Y^(B16)L^(B17)) (AlaPro)^(A-1,0)). As shown in Table 8, theIGF1(Y^(B16)L^(B17))(AlaPro)^(A-1,0) has substantially reduced affinityfor the insulin receptor. Note, based on the data of Table 3, thedipeptide prodrug element lacks the proper structure to allow forspontaneous cleavage of the dipeptide prodrug element, and therefore thedetected insulin receptor binding is not the result of cleavage of theprodrug element.

TABLE 8 Insulin Standard IGF1(YL)_(A-1,0) ^(B16,17)(AlaPro) AVER. STDEVAVER. STDEV. IC₅₀(nM) 0.72 0.09 1.93 .96 % of 100 37.12 Insulin Activity

EXAMPLE 11 Additional IGF Insulin Analogs

Further modifications of the IGF1 (Y^(B16)L^(B17))(YL)B¹⁶B¹⁷ peptidesequence reveal additional IGF insulin analogs that vary in theirpotency at the insulin and IGF-1 receptor. Binding data is presented inTable 9 for each of these analogs (using the assay of Example 3),wherein the position of the modification is designated based on thecorresponding position in the native insulin peptide (DPI=des B26-30).For example, a reference herein to “position B28” absent any furtherelaboration would mean the corresponding position B27 of the B chain ofan insulin analog in which the first amino acid of SEQ ID NO: 2 has beendeleted. Thus a generic reference to “B(Y16)” refers to a substitutionof a tyrosine residue at position 15 of the B chain of the native IGF-1sequence. Data regarding the relative receptor binding of insulin andIGF analogs is provided in Table 9, and data regarding IGF analogstimulated phosphorylation (using the assay of Example 4) is provided inTable 10.

TABLE 9 Receptor Binding Affinity of Insulin and IGF Analogues InsulinReceptor IGF-1 Receptor % % native native % insulin % IGF-1 insulinactivity IGF-1 activity nM (in (0.6 (in (0.55 Analogue IC₅₀: STDev Datetest) nM) IC₅₀: STDev Date test) nM) Ratio IGF-1 A:B 10.41  1.65 Sep. 4,5.8  5.8 2007 IGF-1 A:B(E10Y16L17) 0.66  0.36 May 22, 58.7  90.9  7.85 1.98 Jun. 4,  6.8 7.0  11.9 2007 2007 0.51  0.18 May 29, 98.8 117.6 12.19  2.17 Sep. 18,  5.0 4.5 2007 2007 IGF-1 A:B(E10 Y16L17)-E31E32B—COOH 1.22  0.30 Mar. 20, 36.5  50.0  17.50  2.25 Apr. 4,  3.0 3.1 14.3 2008 2007 IGF-1 A:B(D10Y16L17) DPI A—COOH 0.26  0.02 Nov. 9, 301.0231.0  6.79  1.50 Apr. 4,  7.7 8.1 2007 2008 0.2  0.02 Dec. 4, 380.1300.0 2007 0.42  0.06 Jun. 5, 174.1 144.1 2008 IGF-1 A:B (E10Y16L17) DPI0.38  0.08 Aug. 10, 51.1 157.9  22.89  5.26 Sep. 18,  3.3 2.4  60.2 20072007 IGF-1 A:B (H5D10Y16L17) DPI 0.16  0.07 Nov. 9, 479.0  4.66  0.77Apr. 4, 11.2 11.8  29.1 2007 2008 IGF-1 A:B (H5D10Y16L17) (S═O)DPI 0.25 0.04 Nov. 9, 316.0 2007 IGF-1 A (H8 A9 N21):B(H5D10Y16L17) 0.05  0.01Dec. 4, 1576.7  4.03  0.50 Apr. 4, 12.9 13.6  80.6 DPI A—COOH 2007 20080.09  0.02 Dec. 14, 1667.0 2007 IGF-1 A (H8 A9 N21):B(H5D10Y16L17 A22)0.12  0.02 Dec. 14, 1171.4  22.83  3.53 Apr. 4,  2.3 2.4 190.3 DPIA—COOH 2007 2008 IGF-1 A (H8 A9 N21):B(H5D10Y16L17A22) 0.36  0.10 Dec.14, 400.7 (S═O) DPI A—COOH 2007 IGF-1 A:IGF-1 B(1-8)-In (9-17)-IGF- 1.59 0.62 May 22, 19.1  37.7 131.30 58.05 Jun. 4,  0.3 0.4  82.6 1 B(18-30)2007 2007 IGF-1 A:In (1-17)-IGF-1 B (18-30) 2.77  1.19 May 22, 14.0 21.7  62.50 30.28 Jun. 4,  0.9 0.9  22.6 2007 2007 2.67  0.67 May 18,11.3  22.5 2007 2.48  1.35 May 29, 20.1  24.2 2007 IGF-1 A:InB(1-5)-IGF-1 B(YL)(6-30) 0.31  0.19 Aug. 10, 62.4 193.5  27.54  6.57Sep. 25,  3.6 2  88.8 2007 2007 IGF-2 native  13.33  1.85 Sep. 25,  7.54.5 2007 IGF-2 AB IGF-2 AB(YL) 6.81  3.81 Oct. 10, 8.4  8.8 2007 InA:IGF-1 B(YL) 82.62 31.75 Sep. 4, 0.9  0.7 2007 107.24 65.38 Sep. 4, 0.7 0.6 2007 In A-IGF-2 D:In B-IGF-2 C 0.53  0.11 Sep. 4, 141.0 113.0  1.59 0.34 Sep. 18, 47.6 34.6 2007 2007 0.37  0.05 Oct. 13, 179.1 162.2 14.69  3.02 Sep. 25,  6.8 3.7  39.7 2007 2007 **All C terminals areamides (DPI) unless specified otherwise

TABLE 10 Total Phosphorylation by IGF-1 & IGF-2 Analogues InsulinReceptor IGF-1 Receptor Selctive Analogue EC50: STDev Date % InsulinEC50: STDev Date % IGF Ratio Insulin  1.26 0.098 Dec. 14, 2007 114.8846.66 Jan. 23, 2008 90.89  1.43 0.72 Apr. 1, 2008  86.02 29.35 May 20,2008  1.12 0.11 Mar. 31, 2008  1.53 0.13 Apr. 11, 2008  2.70 0.71 Apr.16, 2008  1.22 0.40 May 20, 2008 IGF-1 54.39 21.102 Dec. 14, 2007 2.3 0.87  0.16 Jan. 23, 2008 100  0.02  0.49  0.13 May 20, 2008  0.97  0.48Jul. 23, 2008 IGF-1 AB IGF-1 A:B(E10Y16L17)  2.57 0.59 Mar. 31, 200849.2  7.42  5.59 Jul. 23, 2008 13 IGF-1 A:B(E10 Y16L17)-E31E32 B—COOH 7.00 2.82 Mar. 31, 2008 18.1  8.52 4.34 Apr. 16, 2008 31.7 IGF-1AB(D10Y16L17) DPI A—COOH  0.08 0.006 Dec. 14, 2007 1575  0.78  0.17 Jan.23, 2008 111.538  9.75  4.38 2.98 Apr. 16, 2008 ?? IGF-1 AB (E10Y16L17)DPI IGF-1 AB (H5D10Y16L17) DPI  12.22  5.46 Jan. 23, 2008 7.1 IGF-1 AB(H5D10Y16L17) (S═O)DPI IGF-1 A (H8 A9 N21) B(H5D10Y16L17)  0.15 0.054Dec. 14, 2007 840  0.43  0.44 Jan. 23, 2008 181.395  2.81 DPI A—COOH 0.25 0.2 Apr. 16, 2008 1080 IGF-1 A (H8 A9 N21) B(H5D10Y16L17A22)  0.350.064 Dec. 14, 2007 360  11.26  2.55 Jan. 23, 2008 7.7 32.54 DPI A—COOH 0.44 0.17 Apr. 16, 2008 614 IGF-1 A (H8 A9 N21)  0.72 0.098 Dec. 14,2007 B(H5D10Y16L17A22) (S═O) DPI A—COOH *All C-terminals are amidesunless specified otherwise.

EXAMPLE 12 Dipeptide Cleavage from Prodrug forms of IGFB16B17 DerivativePeptides

The cleavage of an (pNH2-Phe) amide linked dipeptide AibPro from variousIGF-1 peptides was measured to determine the impact of the peptidesequence or heteroduplex on the edipeptide cleavage. Results for thetested peptides is shown in Table 12 and the data reveals that theIGF1-A chain alone represents a good model for the study of prodrug halflife for IGF1 B:A (YL)^(B16,17) peptides.

TABLE 12 Parent Peptide Half Life (hr)IGF1A(Ala)^(6,11,20)(pNH₂-Phe)^(A19) 2.2IGF1A(Acm)^(6,11,20)(pNH₂-Phe)^(A19) 1.8 IGF1B:A(S—S)^(A7,B7)(Acm)^(A6,11,20,B19)(pNH₂-Phe)^(A19) 1.8 IGF1B:A(pNH₂-Phe)^(A19) 1.6Comparison of prodrug derivatives of the IGF A-chain relative to thedisulfide bound A chain and B chain construct (IGF1 A:B(Y^(B16)L^(B17))revealed the two compounds had similar half lives for the prodrug form.Accordingly, the IGF1A chain alone was determined to be a good model forthe study of pro-drug half life on IGF1 B:A (Y^(B16)L^(B17)). Note theAibAla derivative does not cleave and thus is not a prodrug, but servesto show the modification can inactivate the insulin analog IGF1A:B(Y^(B16)L^(B17))(p-NH₂—F)^(A19)amide. For simplicity, prodrug halflives were determined using only the IGF1 A chain in the absence of theB chain. The half lives of each propeptide was determined as describedin Example 5. The data is presented in Table 13:

TABLE 13 Dipeptide half life on IGF1 dipeptide extended (p-NH₂-F)^(A19)amide Dipeptide Half Life (hr) AiB Pro 2.2 AiBOH Pro 165.0 AiB dPro 1.9AiBOH Sar 2.3 dK(acetyl) Sar 16.3 K Sar 21.8 K(acetyl) N-methyl Ala 23.6dK(acetyl) N-methyl Ala 35.3

The data shows that by altering the substituents on the dipeptideprodrug element that the half life of prodrug can be varied from 2 hrsto >100 hrs.

Additional prodrug derivative peptides were prepared using anIGF1-A(pNH2-F)¹⁹ base peptide and alting the amino acid composition ofthe dipeptide prodrug element linked through the 4-amino phenylalanineat position A19. Dipeptide half lives were measured for differentconstructs both in PBS and in 20% plasma/PBS (i.e. in the presence ofserum enzymes. The results are provided in Table 14. The resultsindicate that three of the four peptides tested were not impacted byserum enzymes.

TABLE 14 Dipeptide half life on IGF1-A(pNH2-F)¹⁹ Half Life (hr) 20% PBSPlasma/PBS AiB Pro 2.2 2.1 AiB dPro 2.1 2.2 AiBOH Sar 2.3 dK N-isobutylGly 4.4 4.1 dK N-hexyl Gly 10.6 dK(acetyl) Sar 17.2 K Sar 21.8 5.9K(acetyl) N-methyl Ala 23.6 dK(acetyl) N-methyl Ala 35.3 AiBOH Pro 165.0K(acetyl) Azetidine-2-carboxylic acid Not cleavable dK(acetyl)Azetidine-2-carboxylic acid Not cleavable

EXAMPLE 13 Receptor Binding of IGF^(B16B17) Derivative Peptides OverTime

Prodrug formulations of IGF^(B16B17) Derivative Peptides were preparedand their degredation over time was measured using the insulin receptorbinding assay of Example 3. Peptides used in the assay were prepared asfollows:

Dipeptide-IGF1A Analogs

If not specified, Boc-chemistry was applied in the synthesis of designedpeptide analogs. Selected dipeptide H₂N-AA1-AA2-COOH was added to(pNH₂-Phe)¹⁹ on IGF1A (Ala)^(6,7,11,20). The IGF-1 A chain C-terminaltripeptide Boc(Fmoc-pNH-Phe)-Ala-Ala was synthesized on MBHA resin.After removal of Fmoc by the treatment with 20% piperidine/DMF at roomtemperature for 30 minutes, Fmoc-AA2 was coupled to the p-amino benzylside chain at A19 by using a threefold excess of amino acid, PyBop, DIEAand catalytic amount of pyridine. The Boc-synthesis of the remainingIGF-1 A chain (Ala)^(6,7,11,20) sequence was completed using an AppliedBiosystems 430A Peptide Synthesizer, yielding IGF-1 A chain(Boc)⁰(Ala)^(6,7,11,20)(Fmoc-AA2-pNH-Phe)¹⁹-MBHA. After the Fmoc groupwas removed from the N-terminus of AA2, Boc-AA1 was then coupled to theamine using threefold excess of amino acid, DEPBT and DIEA. Removal ofthe two Boc groups remaining on the A chain by TFA was followed by HFcleavage, yielding IGF-1 A-chain(Ala)^(6,7,11,20)(H₂N-AA1-AA2-pNH-Phe)¹⁹amide. In the case of AA1 beingd-lysine, acetylation on the ε-amine was performed prior to Boc removal.Dipeptide-IGF-1 A chain analogs were purified by semi-preparativeRP-HPLC and characterized by analytical RP-HPLC and MALDI massspectrometry.

Dipeptide-IGF-1 (YL) Analogs

A selected dipeptide H₂N-AA1-AA2-COOH was added to (pNH₂-Phe)¹⁹ on IGF-1A chain (Acm)^(6,11,20) as described immediately above except PAM resinwas used for the synthesis of IGF-1 A chain to yield a C terminal acidupon HF-cleavage. IGF-1 B chain (YL)^(16,17)(Acm)¹⁹ was synthesized onMBHA resin to yield a C terminal amide. The free thiol on Cys^(B7) wasmodified by Npys through reaction with DTNP at a 1:1 molar ratio in 100%DMSO. Purified dipeptide-IGF-1 A chain and IGF-1 B chain (YL)^(16,17)derivatives were assembled using the “1+2” two step chain combinationstrategy illustrated in Scheme 1. Intermediate and final purificationswere performed on semi-preparative RP-HPLC and characterized byanalytical RP-HPLC and MALDI mass spectrometry.

The IGF^(B16B17) derivative peptide prodrugs were incubated in PBS, pH7.4 at 37° C. and at predetermined time intervals an aliquot was takenand further degredation was quenched with 0.1% TFA and the aliquot wassubjected to analytical HPLC analysis. Peaks a and b, representing theprodrug and active forms of the IGF^(B16B17) derivative peptide wereidentified with LC-MS and quantified by integration of peak area anHPLC. FIGS. 8A-8C show the output of an HPLC analysis of the degradationof the IGF^(B16B17) derivative peptide prodrug:IGF1A(Ala)^(6,7,11,20)(Aib-Pro-pNH—F)¹⁹. Aliquots were taken at 20minutes (FIG. 8A), 81 minutes (FIG. 8B) and 120 minutes (FIG. 8C) afterbeginning the incubation of the prodrug in PBS. The data indicate thespontaneous, non-enzymatic conversion ofIGF1A(Ala)^(6,7,11,20)(Aib-Pro-pNH—F)¹⁹amide toIGF1A(Ala)^(6,7,11,20)(pNH₂—F)¹amide over time.

The degradation of the prodrug forms of IGF^(B16B17) derivative peptidesto there active from was also measured based on the compounds ability tobind to the insulin receptor as measured using the in vitro assay ofExample 3. FIGS. 9A & 9B are graphs depicting the in vitro activity ofthe prodrug Aib,dPro-IGF1YL (dipeptide linked throughout the A194-aminoPhe). FIG. 9A is a graph comparing relative insulin receptorbinding of native insulin (measured at 1 hour at 4° C.) and the A19 IGFprodrug analog (Aib,dPro-IGF1YL) over time (0 hours, 2.5 hours and 10.6hours) incubated in PBS. FIG. 9B is a graph comparing relative insulinreceptor binding of native insulin (measured at 1.5 hour at 4° C.) andthe A19 IGF prodrug analog (Aib,dPro-IGF1YL) over time (0 hours, 1.5hours and 24.8 hours) incubated in 20% plasma/PBS. As indicated by thedata presented in the graph, increased activity is recovered form theA19 IGF prodrug analog sample as the prodrug form is converted to theactive IGF1YL peptide. The activity of the IGF^(B16B17) derivativepeptides was measured relative to insulin receptor binding, and sincethe underlying IGF^(B16B17) derivative peptides have more activity thannative insulin, activity of greater than 100% relative to insulin ispossible.

FIGS. 10A & 10B are graphs depicting the in vitro activity of theprodrug dK,(N-isobutylG)-IGF1YL (dipeptide linked throughout the A194-aminoPhe). FIG. 10A is a graph comparing relative insulin receptorbinding of native insulin (measured at 1 hour at 4° C.) and the A19 IGFprodrug analog (IGF1YL: dK,(N-isobutylG) over time (0 hours, 5 hours and52 hours) incubated in PBS. FIG. 10B is a graph comparing relativeinsulin receptor binding of native insulin (measured at 1.5 hour at 4°C.) and the A19 IGF prodrug analog (IGF1YL: dK,(N-isobutylG) over time(0 hours, 3.6 hours and 24.8 hours) incubated in 20% plasma/PBS. Asindicated by the data presented in the graph, increased activity isrecovered form the A19 IGF prodrug analog sample as the prodrug form isconverted to the active IGF1YL peptide.

FIGS. 11A & 11B are graphs depicting the in vitro activity of theprodrug dK(e-acetyl),Sar)-IGF1YL (dipeptide linked throughout the A194-aminoPhe). FIG. 11A is a graph comparing relative insulin receptorbinding of native insulin (measured at 1 hour at 4° C.) and the A19 IGFprodrug analog (IGF1YL: dK(e-acetyl),Sar) over time (0 hours, 7.2 hoursand 91.6 hours) incubated in PBS. FIG. 11B is a graph comparing relativeinsulin receptor binding of native insulin (measured at 1.5 hour at 4°C.) and the A19 IGF prodrug analog (IGF1YL: dK(e-acetyl),Sar) over time(0 hours, 9 hours and 95 hours) incubated in 20% plasma/PBS. Asindicated by the data presented in the graph, increased activity isrecovered form the A19 IGF prodrug analog sample as the prodrug form isconverted to the active IGF1YL peptide

The invention claimed is:
 1. An insulin prodrug analog comprising an Achain comprising SEQ ID NO: 1, or a modified derivative of SEQ ID NO: 1comprising modification of the amino acid at position A19 to a 4-aminophenylalanine and optionally one or more amino acid substitutions atpositions selected from A5, A8, A9, A10, A14, A15, A17, A18, A19 andA21; a B chain comprising SEQ ID NO: 2, or a modified derivative of SEQID NO: 2 comprising modification of the amino acid at position B16 orB25 to a 4-amino phenylalanine and optionally one or more amino acidsubstitutions at positions selected from B1, B2, B3, B4, B5, B9, B10,B13, B14, B17, B20, B22, B23, B26, B27, B28, B29 and B30 or deletions ofany or all of positions B1-4 and B26-30; and a dipeptide element linkedvia an amide bond to the N-terminal amino group of the A chain or Bchain, or the side chain amino group of an aromatic amine of a4-amino-phenylalanine residue present at position A19, wherein saiddipeptide element comprises an N-terminal C-alkylated amino acidfollowed by an N-alkylated amino acid, wherein the N-alkylated group ofsaid N-alkylated amino acid is a C₁-C₆ alkyl.
 2. An insulin analogcomprising an A chain and B chain, wherein said A chain has the generalstructure of Z-peptide A, wherein peptide A comprises the sequence ofGIVEQCCX₁SICSLYQLENX₂CX₃ (SEQ ID NO: 3), or an analog thereof comprisinga sequence that differs from SEQ ID NO: 3 by 1 to 3 amino acidmodifications, selected from positions A5, A8, A9, A10, A14, A15, A17,A18; and said B chain has the general structure of J-X₁₄-peptide B,wherein peptide B comprises the sequence of X₄LCGX₅X₆LVEALX₇LVCGERGFX₈(SEQ ID NO: 14), or an analog thereof comprising a sequence that differsfrom SEQ ID NO: 14 sequence by 1 to 3 amino acid modifications, selectedfrom positions B5, B13, B14, B17, B20, B22, and B23 said B chain beinglinked to said A chain through intermolecular disulfide linkages andoptionally through an amide bond linkage between the carboxy terminus ofthe B chain and the amino terminus of the A chain to form a single chainpolypeptide wherein Z and J are independently H or a dipeptide elementof the structure U-O, wherein U-O is linked to SEQ ID NO: 3 or SEQ IDNO: 14 through an amide bond, further wherein U-O is a compound havingthe general structure of Formula I:

wherein R₁, R₂, R₄ and R₈ are independently selected from the groupconsisting of H, C₁-C₁₈ alkyl, C₂-C₁₈ alkenyl, (C₁-C₁₈ alkyl)OH, (C₁-C₁₈alkyl)SH, (C₂-C₃ alkyl)SCH₃, (C₁-C₄ alkyl)CONH₂, (C₁-C₄ alkyl)COOH,(C₁-C₄ alkyl)NH₂, (C₁-C₄ alkyl)NHC(NH₂ ⁺)NH₂, (C₀-C₄ alkyl)(C₃-C₆cycloalkyl), (C₀-C₄ alkyl)(C₂-C₅ heterocyclic), (C₀-C₄ alkyl)(C₆-C₁₀aryl)R₇, (C₁-C₄ alkyl)(C₃-C₉ heteroaryl), and C₁-C₁₂ alkyl(W₁)C₁-C₁₂alkyl, wherein W₁ is a heteroatom selected from the group consisting ofN, S and O, or R₁ and R₂ together with the atoms to which they areattached form a C₃-C₁₂ cycloalkyl; or R₄ and R₈ together with the atomsto which they are attached form a C₃-C₆ cycloalkyl; R₃ is selected fromthe group consisting of C₁-C₁₈ alkyl, (C₁-C₁₈ alkyl)OH, (C₁-C₁₈alkyl)NH₂, (C₁-C₁₈ alkyl)SH, (C₀-C₄ alkyl)(C₃-C₆)cycloalkyl, (C₀-C₄alkyl)(C₂-C₅ heterocyclic), (C₀-C₄ alkyl)(C₆-C₁₀ aryl)R₇, and (C₁-C₄alkyl)(C₃-C₉ heteroaryl) or R₄ and R₃ together with the atoms to whichthey are attached form a pyrrolidine ring; R₅ is NHR₆ or OH; R₆ is H,C₁-C₈ alkyl or R₆ and R₁ together with the atoms to which they areattached form a 4, 5 or 6 member heterocyclic ring; and R₇ is selectedfrom the group consisting of hydrogen, C₁-C₁₈ alkyl, C₂-C₁₈ alkenyl,(C₀-C₄ alkyl)CONH₂, (C₀-C₄ alkyl)COOH, (C₀-C₄ alkyl)NH₂, (C₀-C₄alkyl)OH, and halo; X₁ is selected from the group consisting ofthreonine, histidine and lysine; X₂ is an amino acid of the generalstructure

wherein m is an integer selected from the group consisting of 0-3; X isselected from the group consisting of OH, OCH₃, NH₂ and NHR₁₀, whereinR₁₀ is a dipeptide element comprising the general structure: U-O; X₃ isselected from the group consisting of asparagine, glycine and alanine;X₄ is selected from the group consisting of histidine and threonine; X₅is selected from the group consisting of alanine, glycine and serine; X₆is selected from the group consisting of histidine, aspartic acid,glutamic acid, homocysteic acid and cysteic acid; X₇ is an amino acid ofthe general structure

wherein m is an integer selected from 0-3 and X₁₂ is selected from thegroup consisting of OH, NH₂, NHR₁₁ and OCH₃, wherein R₁₁ is a dipeptideelement comprising the general structure: U-O; X₈ is an amino acid ofthe general structure

wherein m is an integer selected from 0-3 and X₁₃ is selected from thegroup consisting of H, OH, NH₂, NHR₁₂ and OCH₃, wherein R₁₂ is adipeptide element comprising the general structure: U-O; X₁₄ is selectedfrom the group consisting of a bond, X₉VNQ (SEQ ID NO: 21), VNQ, NQ andQ, wherein X₉ is selected from the group consisting of phenylalanine anddesamino-phenylalanine; with the proviso that one or more of X, X₁₂,X₁₃, J and Z is a dipeptide element comprising the general structure:U-O, and R₁ and R₂ are not both H.
 3. The insulin analog of claim 2wherein said A-peptide comprises the sequence of GIVEQCCTSICSLYQLENYCN(SEQ ID NO: 1), said X₁₄-peptide B comprises the sequence ofFVNQHLCGSHLVEALYLVCGERGFFYTPKT (SEQ ID NO: 2) orFVNQHLCGSHLVEALYLVCGERGFFYTKPT (SEQ ID NO: 9); Z is H; and J is acompound having the general structure of Formula I:

wherein R₁ is selected from the group consisting of H and C₁-C₄ alkyl;R₂ is selected from the group consisting of H, C₁-C₆ alkyl, C₂-C₈alkenyl, (C₁-C₄ alkyl)OH, (C₁-C₄ alkyl)NH₂, (C₀-C₄ alkyl)(C₃-C₆cycloalkyl), (C₀-C₄ alkyl)(C₆-C₁₀ aryl)R₇, and CH₂(C₅-C₉ heteroaryl) orR₂ and R₆ together with the atoms to which they are attached form a 5member heterocyclic ring; R₃ is C₁-C₆ alkyl; R₄ is selected from thegroup consisting of H and C₁-C₄ alkyl; and R₈ is H.
 4. The insulinanalog of claim 2, wherein Z is a dipeptide element comprising thegeneral structure:

wherein R₁ and R₂ are independently C₁-C₁₈ alkyl or aryl; or R₁ and R₂are linked through —(CH₂)_(p)—, wherein p is 2-9; R₃ is C₁-C₁₈ alkyl; R₄and R₈ are each hydrogen; and R₅ is an amine.
 5. The insulin analog ofclaim 2, wherein X is NHR₁₀; R₁ and R₂ are independently C₁-C₁₈ alkyl oraryl; R₃ is C₁-C₁₈ alkyl or R₃ and R₄ together with the atoms to whichthey are attached form a 4-12 heterocyclic ring; R₄ and R₈ areindependently selected from the group consisting of hydrogen, C₁-C₁₈alkyl and aryl; and R₅ is an amine or a hydroxyl.
 6. The insulin analogof claim 2, wherein X is NHR₁₀; R₁ is selected from the group consistingof hydrogen, C₁-C₁₈ alkyl and aryl, or R₁ and R₂ are linked through—(CH₂)_(p)—, wherein p is 2-9; R₃ is C₁-C₁₈ alkyl or R₃ and R₄ togetherwith the atoms to which they are attached form a heterocyclicpyrrolidine ring; R₄ and R₈ are independently selected from the groupconsisting of hydrogen, C₁-C₁₈ alkyl and aryl; and R₅ is an amine orN-substituted amine.
 7. The insulin analog of claim 2, wherein saidpeptide A comprises the sequence of GIVEQCCX₁SICSLYQLENX₂CX₃ (SEQ ID NO:3) and said peptide B comprises the sequence of X₄LCGX₅X₆LVEALYLVCGERGFF(SEQ ID NO: 4) wherein Z is H; J is a dipeptide element comprising thegeneral structure:

X₁ is selected from the group consisting of threonine and histidine; X₂is an amino acid of the general structure

wherein X is selected from the group consisting of OH, NH₂ and OCH₃, X₃is selected from the group consisting of asparagine and glycine; X₄ isselected from the group consisting of histidine and threonine; X₅ isselected from the group consisting of alanine, glycine and serine; X₆ isselected from the group consisting of histidine, aspartic acid, glutamicacid, homocysteic acid and cysteic acid; R₁, R₂, R₄ and R₈ areindependently selected from the group consisting of H, C₁-C₁₈ alkyl,C₂-C₁₈ alkenyl, (C₁-C₁₈ alkyl)OH, (C₁-C₁₈ alkyl)SH, (C₂-C₃ alkyl)SCH₃,(C₁-C₄ alkyl)CONH₂, (C₁-C₄ alkyl)COOH, (C₁-C₄ alkyl)NH₂, (C₁-C₄alkyl)NHC(NH₂ ⁺)NH₂, (C₀-C₄ alkyl)(C₃-C₆ cycloalkyl), (C₀-C₄alkyl)(C₂-C₅ heterocyclic), (C₀-C₄ alkyl)(C₆-C₁₀ aryl)R₇, (C₁-C₄alkyl)(C₃-C₉ heteroaryl), and C₁-C₁₂ alkyl(W)C₁-C₁₂ alkyl, wherein W isa heteroatom selected from the group consisting of N, S and O, or R₁ andR₂ together with the atoms to which they are attached form a C₃-C₁₂cycloalkyl or aryl; or R₄ and R₈ together with the atoms to which theyare attached form a C₃-C₆ cycloalkyl; R₃ is selected from the groupconsisting of C₁-C₁₈ alkyl, (C₁-C₁₈ alkyl)OH, (C₁-C₁₈ alkyl)NH₂, (C₁-C₁₈alkyl)SH, (C₀-C₄ alkyl)(C₃-C₆)cycloalkyl, (C₀-C₄ alkyl)(C₂-C₅heterocyclic), (C₀-C₄ alkyl)(C₆-C₁₀ aryl)R₇, and (C₁-C₄ alkyl)(C₃-C₉heteroaryl); R₅ is NHR₆ or OH; R₆ is H, C₁-C₈ alkyl or R₆ and R₂together with the atoms to which they are attached form a 4, 5 or 6member heterocyclic ring; and R₇ is selected from the group consistingof H and OH, with the proviso that when R₄ and R₃ together with theatoms to which they are attached form a 5 or 6 member heterocyclic ring,then R₁ and R₂ are other than H.
 8. The insulin analog of claim 7wherein X₁₄-peptide B comprises the sequenceX₉VNQX₄LCGX₅X₆LVEALYLVCGERGFFYTPKT (SEQ ID NO: 12) orX₉VNQX₄LCGX₅X₆LVEALYLVCGERGFFYTKPT (SEQ ID NO: 13) wherein X₄ isselected from the group consisting of histidine and threonine; X₅ isselected from the group consisting of alanine, glycine and serine; X₆ isselected from the group consisting of histidine, aspartic acid, glutamicacid, homocysteic acid and cysteic acid; and X₉ is phenylalanine.
 9. Theinsulin analog of claim 8 wherein R₁ is selected from the groupconsisting of H and C₁-C₄ alkyl; R₂ is selected from the groupconsisting of H, C₁-C₆ alkyl, C₂-C₈ alkenyl, (C₁-C₄ alkyl)OH, (C₁-C₄alkyl)NH₂, (C₀-C₄ alkyl)(C₃-C₆ cycloalkyl), (C₀-C₄ alkyl)(C₆-C₁₀aryl)R₇, and CH₂(C₅-C₉ heteroaryl); R₃ is C₁-C₆ alkyl; R₄ is selectedfrom the group consisting of H and C₁-C₄ alkyl.
 10. The insulin analogof claim 9 wherein R₃ is CH₃ and R₄ is H; and R₅ is NHR₆.
 11. Theinsulin analog of claim 7 wherein X₄ is histidine; X₅ is serine; X₆ ishistidine; R₃ is C₁-C₆ alkyl; and R₄ is selected from the groupconsisting of H and C₁-C₄ alkyl.
 12. The insulin analog of claim 11wherein R₃ is methyl; R₄ is selected from the group consisting of H andC₁-C₄ alkyl.
 13. The insulin analog of claim 7 wherein a polyethyleneglycol chain is linked to the insulin analog.
 14. An insulin analogcomprising an A chain and a B chain wherein said A chain has the generalstructure Z-peptide A, wherein peptide A comprises the sequence ofGIVEQCCX₁SICSLYQLENX₂CX₃ (SEQ ID NO: 3), or an analog thereof comprisinga sequence that differs from SEQ ID NO: 3 by 1 to 3 amino acidmodifications, selected from positions A5, A8, A9, A10, A14, A15, A17,A18; and said B chain has the general structure J-X₁₄-peptide B, whereinpeptide B comprises the sequence of X₄LCGX₅X₆LVEALX₇LVCGERGFX₈ (SEQ IDNO: 14) or a sequence that differs from SEQ ID NO: 14 by 1 to 3 aminoacid modifications, selected from positions B5, B13, B14, B17, B20, B22,and B23, said B chain being linked to said A chain throughintermolecular disulfide linkages and optionally through an amide bondlinkage between the carboxy terminus of the B chain and the aminoterminus of the A chain to form a single chain polypeptide, wherein Zand J are independently H or a dipeptide element comprising the generalstructure:

X₁₄ is selected from the group consisting of a bond, X₉VNQ (SEQ ID NO:21), VNQ, NQ and Q; X₁ is selected from the group consisting ofthreonine and histidine; X₂ is an amino acid of the general structure

wherein X is selected from the group consisting of OH, NHR₁₀ and OCH₃,wherein R₁₀ is H or a dipeptide element comprising the generalstructure:

X₃ is selected from the group consisting of asparagine, glycine andalanine; X₄ is selected from the group consisting of histidine andthreonine and serine; X₅ is selected from the group consisting ofalanine, glycine and serine; X₆ is selected from the group consisting ofhistidine, aspartic acid, glutamic acid, homocysteic acid and cysteicacid; X₇ is an amino acid of the general structure

wherein X₁₂ is selected from the group consisting of OH, OCH₃ and NHR₁₁,wherein R₁₁ is H or a dipeptide element comprising the generalstructure:

X₈ is an amino acid of the general structure

wherein X₁₃ is selected from the group consisting of H, OH, OCH₃ andNHR₁₂, wherein R₁₂ is H or a dipeptide element comprising the generalstructure:

X₉ is selected from the group consisting of phenylalanine anddesamino-phenylalanine; wherein R₁ is selected from the group consistingof H and C₁-C₈ alkyl; and R₂ and R₄ are independently selected from thegroup consisting of H, C₁-C₈ alkyl, C₂-C₈ alkenyl, (C₁-C₄ alkyl)OH,(C₁-C₄ alkyl)SH, (C₂-C₃ alkyl)SCH₃, (C₁-C₄ alkyl)CONH₂, (C₁-C₄alkyl)COOH, (C₁-C₄ alkyl)NH₂, (C₁-C₄ alkyl)NHC(NH₂ ⁺) NH₂, (C₀-C₄alkyl)(C₃-C₆ cycloalkyl), (C₀-C₄ alkyl)(C₆-C₁₀ aryl)R₇, and CH₂(C₅-C₉heteroaryl); R₃ is selected from the group consisting of C₁-C₈ alkyl,(C₁-C₄ alkyl)OH, (C₁-C₄ alkyl)SH, (C₁-C₄ alkyl)NH₂, (C₃-C₆)cycloalkyl;R₅ is NHR₆ or OH; R₆ is H, or R₆ and R₂ together with the atoms to whichthey are attached form a 5 or 6 member heterocyclic ring; and R₇ isselected from the group consisting of H and OH, with the proviso thatone and only one of R₁₀, R₁₁, R₁₂, J and Z comprises a dipeptide elementof the general structure:


15. The insulin analog of claim 14 wherein Z or J comprises a dipeptideelement of the general structure:

and said dipeptide element is i) acylated with an acyl group comprising16 to 30 carbon atoms; ii) pegylated with one or two polyethylene glycolchains wherein the combined molecular weight of the polyethylene glycolchains ranges from about 20,000 to about 80,000 Daltons; or iii)alkylated with an alkyl group comprising 8 to 20 carbon atoms; or iv)said dipeptide element is acylated with an acyl group comprising 16 to30 carbon atoms and pegylated with one or two polyethylene glycol chainswherein the combined molecular weight of the polyethylene glycol chainsranges from about 20,000 to about 80,000 Daltons.
 16. The insulin analogof claim 14 wherein a polyethylene glycol chain or a sequesteringmacromolecule is covalently linked to the R₂ side chain of the dipeptideelement comprising the general structure:

wherein R₂ represents (C₁-C₄ alkyl)OH, (C₁-C₄ alkyl)SH, or (C₁-C₄alkyl)NH₂.
 17. An insulin analog comprising an A chain and B chain,wherein said A chain has the general structure Z-peptide A, whereinpeptide A comprises the sequence of GIVEQCCX₁SICSLYQLENX₂CX₃ (SEQ ID NO:3), or an analog thereof comprising a sequence that differs from SEQ IDNO: 3 by 1 to 3 amino acid modifications, selected from positions A5,A8, A9, A10, A14, A15, A17, A18; and said B chain has the generalstructure J-X₁₄-peptide B, wherein peptide B comprises the sequence ofX₄LCGX₅X₆LVEALX₇LVCGERGFX₈ (SEQ ID NO: 14), or an analog thereofcomprising a sequence that differs from SEQ ID NO: 14 sequence by 1 to 3amino acid modifications, selected from positions B5, B13, B14, B17,B20, B22, and B23 said B chain being linked to said A chain throughintermolecular disulfide linkages and optionally through an amide bondlinkage between the carboxy terminus of the B chain and the aminoterminus of the A chain to form a single chain polypeptide wherein Z andJ are independently H or a dipeptide element of the structure U-O,wherein U is aminoisobutyric acid, hydroxyl aminoisobutyric acid, anamino acid in the D-stereochemical configuration or a hydroxyl acid inthe D-stereochemical configuration, and O is an N-alkylated amino acid,and O is linked to SEQ ID NO: 3 or SEQ ID NO: 14 through an amide bondlinkage; X₁ is selected from the group consisting of threonine,histidine and lysine; X₂ is an amino acid of the general structure

wherein m is an integer selected from the group consisting of 0-3; X isselected from the group consisting of OH, OCH₃, NH₂ and NHR₁₀, whereinR₁₀ is a dipeptide element comprising the general structure: U-O; X₃ isselected from the group consisting of asparagine, glycine and alanine;X₄ is selected from the group consisting of histidine and threonine; X₅is selected from the group consisting of alanine, glycine and serine; X₆is selected from the group consisting of histidine, aspartic acid,glutamic acid, homocysteic acid and cysteic acid; X₇ is an amino acid ofthe general structure

wherein m is an integer selected from 0-3 and X₁₂ is selected from thegroup consisting of OH, NH₂, NHR₁₁ and OCH₃, wherein R₁₁ is a dipeptideelement comprising the general structure: U-O; X₈ is an amino acid ofthe general structure

wherein m is an integer selected from 0-3 and X₁₃ is selected from thegroup consisting of H, OH, NH₂, NHR₁₂ and OCH₃, wherein R₁₂ is adipeptide element comprising the general structure: U-O; X₁₄ is selectedfrom the group consisting of a bond, X₉VNQ (SEQ ID NO: 21), VNQ, NQ andQ, wherein X₉ is selected from the group consisting of phenylalanine anddesamino-phenylalanine; with the proviso that one or more of X, X₁₂,X₁₃, J and Z is a dipeptide element comprising the general structure:U-O, and R₁ and R₂ are not both H.
 18. A pharmaceutical compositioncomprising the insulin analog of claim 2, and a pharmaceuticallyacceptable carrier.
 19. A method of treating diabetes, said methodcomprising administering an effective amount of a pharmaceuticalcomposition of claim
 18. 20. The insulin analog of claim 15 wherein saidpeptide A comprises the sequence of GIVEQCCX₁SICSLYQLENX₂CX₃ (SEQ ID NO:3) and said peptide B comprises the sequence of X₄LCGX₅X₆LVEALX₇LVCGERGFX₈ (SEQ ID NO: 14), wherein X₁ is histidine or threonine; X₂ and X₇are both tyrosine; X₃ is asparagine; X₄ is histidine; X₅ is serine; X₆is histidine; X₈ is phenylalanine or tyrosine; and X₁₄ is selected fromthe group consisting of a bond, FVNQ (SEQ ID NO: 21), VNQ, NQ and Q. 21.The insulin analog of claim 20 wherein said A chain comprises thesequence GIVEQCCTSICSLYQLENYCN (SEQ ID NO: 1), and said X₁₄-peptide Bcomprises the sequence of FVNQHLCGSHLVEALYLVCGERGFFYTPKT (SEQ ID NO: 2)or FVNQHLCGSHLVEALYLVCGERGFFYTKPT (SEQ ID NO: 9).
 22. The insulin analogof claim 20 wherein R₁ and R₂ are independently selected from the groupconsisting of hydrogen, C₁-C₈ alkyl and (C₁-C₄ alkyl)NH₂, or R₁ and R₂are linked through (CH₂)_(p), wherein p is 2-9; R₃ is C₁-C₈ alkyl; R₄ isselected from the group consisting of hydrogen and C₁-C₈ alkyl; and R₅is NH₂; with the proviso that both R₁ and R₂ are not hydrogen.
 23. Theinsulin analog of claim 21 wherein the dipeptide prodrug elementcomprises the general structure:

wherein R₂ comprises (C₁-C₄ alkyl)NH₂ that is acylated at the aminogroup of R₂ with an acyl group of sufficient size to bind serum albumin;R₁ and R₄ are each hydrogen; R₃ is CH₃; and R₅ is NH₂.
 24. The insulinanalog of claim 23 wherein the lysine of the dipeptide prodrug elementis in the D-stereochemical configuration and said acyl group iscovalently linked to the amino group of the lysine side chain.
 25. Apharmaceutical composition comprising the insulin analog of claim 20,and a pharmaceutically acceptable carrier.
 26. The insulin analog ofclaim 23 wherein said dipeptide element is acylated with an acyl groupcomprising 16 to 30 carbon atoms.
 27. A dimer or multimer comprising theinsulin analog of claim 21.